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Raquel Anderson: Como saber se estamos prestes a fazer uma fofoca? Nancy DeMoss Wolgemuth tem um...

Neću otkriti toplu vodu kada kažem da živimo brzim tempom i kada sedneš sa prijateljima na kafu - razmenite hajlajtove svog života i idete dalje. Zato i ne čudi da je podkast kao žanr doživeo bum. Ljudi žele da slušaju razgovore natenane, bez međusobnih prekidanja i tenzija. Tako je i nastao serijal ŠBB KBB - "Šta bi bilo kad bilo" - pa nevidljim slovima u nastavku stoji - da se ljudski ispričamo. Gost nulte epizode je Boško Jakovljević, TV lice, model, modni kreator, ali pre svega moj prijatelj koga znam još iz osnovne škole. Oboje smo prošli iste istorijske događaje i imamo puno iskustva kada su u pitanju društveni fenomeni i život ovog grada. Jedna od tema je upravo i život u dva veka i dva milenijuma. Da li je to prednost ili hendikep. Pričamo i o fenomenima svakodnevice, algoritmima koji su nam se uselili u život. A tu je priča o tome šta se desi kada spoznaš da si u nekim životnim situacijama ispao budala, o odrastanju i sazrevanju u javnosti, pred kamerama i objektivima foto aparata. O napadima panike, ali i o tome kako izabrati bicikl i patike, kao i o tome kako su masti važne za mozak i šta je bila idealna užina sedamdesetih godina 20. veka. Posebna delicija je segment u kome predlažemo koga da guglujete, kao i u kojim ponudama kulturnog života možemo uživati ove jeseni. Slušamo se i gledamo nedeljom u 18h.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.31.522373v1?rss=1 Authors: Coly, P.-M., Chatterjee, S., Mezine, F., El Jekmek, C., Devue, C., Nipoti, T., Lara Corona, M., Dingli, F., Loew, D., van Niel, G., Boulanger, C. M. Abstract: Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Conversely, high shear stress (HSS) has atheroprotective effects on the endothelium. Endothelial cell-derived extracellular vesicles have been shown to regulate inflammation, senescence and angiogenesis and therefore play a crucial role in vascular homeostasis and disease. While previous studies have shown links between hemodynamic forces and extracellular vesicle release, the exact consequences of shear stress on the release and uptake of endothelial EVs remains elusive. Our aim is therefore to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress. Confluent human umbilical vein endothelial cells (HUVEC) were exposed to either LSS or HSS for 24 hours. Large and small EVs were isolated from conditioned medium by sequential centrifugation and size exclusion chromatography. They were characterized by TEM, Western blot analysis of EV markers, tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using fluorescently-labeled EVs and differences between groups were assessed by flow cytometry and confocal microscopy. We found that levels of large and small EVs in HUVEC conditioned media were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions compared to HSS. Additionally, endothelial LSS-EVs appeared to have a greater affinity for HUVECs than HSS-EVs or EVs derived from platelets, red blood cells, granulocytes and peripheral blood mononuclear cells. Proteomic analysis revealed that LSS-EVs were enriched in adhesion proteins such as PECAM1, MCAM, which were involved in EV uptake by endothelial cells. LSS-EVs also carried mitochondrial material, which may be involved in elevating reactive oxygen species levels in recipient cells. These findings suggest that endothelial shear stress has a significant impact during EV biogenesis and uptake. Given the major role of EVs and shear stress in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

On Episode 15 of the Stroke Alert Podcast, host Dr. Negar Asdaghi highlights two articles from the April 2022 issue of Stroke: “Kawasaki Disease May Increase the Risk of Subsequent Cerebrovascular Disease” and “Effect of Moderate and Severe Persistent Hyperglycemia on Outcomes in Patients With Intracerebral Hemorrhage.” She also interviews Dr. François Gros-Louis about his article “Moyamoya Disease Susceptibility Gene RNF213 Regulates Endothelial Barrier Function.” Dr. Negar Asdaghi:                        1) How would you counsel the parent of a child who has just recovered from Kawasaki disease on their child's future risk of having a stroke? 2) Should we or should we not treat stress hyperglycemia in the setting of acute intracerebral hemorrhage? 3) What is the CRISPR-Cas9 gene editing technology? And why, if you haven't heard of it already, you most definitely should be listening to this podcast? We're back here with the April issue of the Stroke Alert Podcast, and this is the latest in Stroke. Stay with us. Dr. Negar Asdaghi:                        Welcome back to another extremely informative Stroke Alert Podcast. My name is Negar Asdaghi. I'm an Associate Professor of Neurology at the University of Miami Miller School of Medicine, and your host for the monthly Stroke Alert Podcast. The April 2022 issue of Stroke contains a range of really exciting papers and topics. In the paper titled "Vascular Response to Spreading Depolarization Predicts Stroke Outcome," we have a really interesting in vivo mouse model of ischemic stroke, looking at the spreading patterns of cortical depolarization and the subsequent vascular response to this by way of hyperemia. The researchers from Zurich University, led by Dr. Binder and colleagues, walk us through how the patterns of hyperemia can actually predict the severity of subsequent ischemic injury. Dr. Negar Asdaghi:                        In a separate paper in this issue of the journal, we're reminded of how the classic NIH Stroke Scale can underestimate the severity of neurological symptoms and outcomes in patients with posterior circulation infarcts. In the paper led by Dr. Alemseged and colleagues, the investigators from the Royal Melbourne Hospital in Australia evaluate the prognostic accuracy of the Posterior NIH Stroke Scale, which is the modified version of the classic NIH Stroke Scale, in predicting the outcomes of patients with posterior circulation infarcts. Dr. Negar Asdaghi:                        I encourage you to review these papers in addition to listening to our podcast today. Later in the podcast, I have the great pleasure of interviewing Dr. François Gros-Louis from Laval University in Quebec to discuss the latest in gene editing technology and how this technology has helped his team unravel the biological function of RNF213 susceptibility gene in Moyamoya disease. But first with these two articles. Dr. Negar Asdaghi:                        Kawasaki disease, which was first described in 1976, is an acute febrile illness predominantly affecting children younger than five years of age. In addition to fever, other clinical signs of the disease include rash, bilateral conjunctival injection, cervical lymphadenopathy, swelling of the hands and feet, and irritation and inflammation of the mouth, lips, and throat. Now, for those of us like me who are adult neurologists, here is a quick review of the pathophysiology of Kawasaki disease. Dr. Negar Asdaghi:                        This is a medium vessel vasculopathy, most significantly affecting the coronary arteries, a vasculopathy that is characterized by three linked pathological processes, necrotizing arteritis, subacute to chronic vasculitis, and luminal myofibroblastic proliferation. So, simply put, these processes can lead to stenotic lesions in various vascular beds, which are affected by this disease. Dr. Negar Asdaghi:                        And as we mentioned earlier, the most recognized vascular blood vessels affected by Kawasaki disease are the coronary arteries, which can lead to myocardial ischemia, infarction, and sudden death in these cases. However, involvement of other vascular beds, including cerebral vessels, are also increasingly reported as part of Kawasaki disease. Dr. Negar Asdaghi:                        So, in the current issue of the journal, Dr. Chien-Heng Lin from the Division of Pediatric Pulmonology at China Medical University Children's Hospital in Taiwan and colleagues studied the subsequent risk of cerebrovascular events in patients with Kawasaki disease. Using the National Health Insurance Research Database of Taiwan, they collected data on 8467 children with Kawasaki disease from 2000 to 2012. And for each child with Kawasaki, data was also collected on four randomly selected non-Kawasaki disease children who were matched with the Kawasaki cohort for sex, urbanization level of residence, and parental occupation. Dr. Negar Asdaghi:                        So, that gave them a sample size of over 33,000 children for their non-Kawasaki cohort. And then they compared the risk of subsequent stroke in children between the two cohorts. The study period for any given patient would end when the said patient was either diagnosed with a cerebrovascular disease or withdrew from the research database. Dr. Negar Asdaghi:                        So, in terms of their demographics, 61% of patients in the Kawasaki group were boys; 88% of the Kawasaki cohort were younger than five years of age. So, here are the findings. Number one, the incident rate of subsequent cerebrovascular disease was 14.7 per hundred thousand person years in the Kawasaki cohort versus only 4.6 per hundred thousand person years in the non-Kawasaki cohort. That's greater than a threefold higher incidence rate of cerebrovascular disorders for children who had Kawasaki disease before. Dr. Negar Asdaghi:                        This finding was independent of other potential confounders, which they adjusted for in their multivariate analysis. Now, the length of follow up was a median of 9.8 years for the entire cohort. And on the issue of time, they found two important associations. The first finding was that when the follow-up time was stratified by five-year periods, Kawasaki disease cohort patients showed a significantly higher risk of developing a stroke within the first five years after being diagnosed. Dr. Negar Asdaghi:                        And the second important association was that when they looked at the age at the time of diagnosis of Kawasaki, children who were younger than five years at the time of diagnosis were at a significantly higher risk of having a future stroke as compared to those who were older than five at the time of diagnosis. Dr. Negar Asdaghi:                        So, simply put, the risk of subsequent stroke was higher in children who acquired the disease at a younger age, and that risk was higher in the first few years after the diagnosis of Kawasaki disease. The authors discuss a number of putative mechanisms to link Kawasaki with stroke. The most important being a cardiac source of embolism that we already alluded to, but other etiologies, including medium vessel cerebral vasculitis, or hypercoagulability in the setting of increased systemic inflammation, and even Kawasaki disease-associated aneurysmal rupture to cause hemorrhagic forms of stroke, are discussed in the paper and should be considered in the correct setting in children with a prior history of this disease. Dr. Negar Asdaghi:                        So, what we learned from this large population-based pediatric study is that Kawasaki disease does indeed increase the risk of subsequent cerebrovascular disorders, and its influence is stronger in children who are diagnosed with this condition under the age of five, and the time period during which the risk of stroke is the highest is within the first five years after the diagnosis. Dr. Negar Asdaghi:                        In the setting of spontaneous intracerebral hemorrhage, or ICH, much research has focused on the association between hypertension and blood pressure-lowering therapies and hematoma expansion and functional outcomes, but a lot less attention relatively has been given to the impact of hyperglycemia and ICH-related outcomes. Dr. Negar Asdaghi:                        The current guidelines state that serum glucose should be monitored and both hypo- and hyperglycemia should be avoided in the setting of ICH. The older studies have given us inconsistent results as to whether or not hyperglycemia can increase the risk of ICH-related mortality. More recent studies have suggested that perhaps persistent hyperglycemia is indeed a predictor of poor neurological outcomes in ICH, but these results come from smaller single-center studies, which require further confirmation. And this confirmation is exactly what Dr. Adnan Qureshi from Zeenat Qureshi Stroke Institute and the Department of Neurology at University of Missouri and colleagues aim to give us in their study titled "Effect of Moderate and Severe Persistent Hyperglycemia on Outcomes in Patients With Intracerebral Hemorrhage." Dr. Negar Asdaghi:                        So, they use data from the ATACH-2 study, and a quick reminder that ATACH-2 was a multicenter randomized control trial of a thousand patients with acute spontaneous intracerebral hemorrhage enrolled within four and a half hours from symptom onset, and patients were randomized to either the intensive blood pressure control treatment arm to maintain their systolic blood pressure goal of 110 to 139 millimeter of mercury versus standard treatment arm, which was keeping their systolic blood pressure above 140, between 140 to 179 millimeter of mercury, in the first 24 hours after randomization. Dr. Negar Asdaghi:                        You will recall that enrollment of ATACH-2 was stopped early because of futility after pre-specified interim analysis. The main results of the trial was published in 2016 in New England Journal of Medicine, and the primary results did not show a lower rate of death or disability in patients assigned to the intensive treatment group. Dr. Negar Asdaghi:                        So, in the current paper, in this current issue of the journal, the authors looked at the glycemic status of the patients enrolled in the trial. As part of the trial, patients had a complete chemistry panel at baseline, 24, 48, and 72 hours from onset. So, they used the glucose measurement from this panel and defined moderate hyperglycemia as serum glucose level of over 140 and under 180 and severe hyperglycemia as serum glucose levels of equal or greater than 180. Dr. Negar Asdaghi:                        Now, persistent hyperglycemia was if two consecutive serum glucose levels were in the moderate or severe categories. And, very simply, they looked at the effects of hyperglycemia on ICH outcomes. And importantly, they evaluated whether hyperglycemia modified the effects of intensive blood pressure reduction on outcomes of ICH. So, of the thousand participants in ATACH-2, 11% had persistent moderate hyperglycemia, and 17% had severe persistent hyperglycemia. Those in the hyperglycemic group were more likely to be diabetic, not surprisingly, more likely to have a history of hypertension and dyslipidemia as compared to the normal glycemic patients. Dr. Negar Asdaghi:                        And here are the results. Number one, serious adverse events were higher in the hyperglycemic groups, whether we're talking about the moderate or the severe hyperglycemic groups. This is despite the fact that the rate of hematoma expansion and perihematomal edema was not different based on the hyperglycemic status. However, the hyperglycemic patients were more likely to have serious adverse events, which were operationally defined as complications that were not expected to have occurred from the study intervention, in this case, the intensive hypertensive therapy, and resulted in either death or prolonged hospitalization or persistent or significant disabilities. Now, serious renal adverse events, which are, of course, expected as a complication for aggressive blood pressure therapy, were also significantly higher in the hyperglycemic category. Dr. Negar Asdaghi:                        Now, their next important finding was that overall, both moderate and severe hyperglycemia was associated with higher odds of 90 days disability or death post-ICH adjusting for typical variables that could predict these outcomes, such as GCS score, hematoma volume, presence or absence of intraventricular hemorrhage, amongst other factors that they accounted for. Dr. Negar Asdaghi:                        Now, number three, this is perhaps the most important finding of the study. Among patients without a preexisting history of diabetes, both moderate and severe hyperglycemia increased the risk of death and disability at 90 days after adjusting for all the potential confounders, but hyperglycemia was not associated with these poor outcomes in those with a prior history of diabetes. I'm going to pause here to let this information sink in. Let's go over them again, stress hyperglycemia in non-diabetics was associated with poor ICH outcomes, but high sugars in diabetics did not predict the same poor outcomes. And finally, they looked at the possible interactions between the glycemic status and the ATACH-2 intervention, which as we alluded to earlier, which was intensive versus standard blood pressure therapy, and it turns out that the intensive systolic blood pressure reduction was indeed associated with a lower rate of hematoma expansion only in patients with normal glycemia, but not in those with moderate or severe hyperglycemia. Dr. Negar Asdaghi:                        So, this is again food for thought. Simply put, if the sugars are not well controlled, it appears that intensive blood pressure control would not lower the rate of hematoma expansion. Blood pressure lowering works when the sugar levels are controlled. So, overall, here are the two simple messages of this study. Number one, hyperglycemia in the acute setting of intracerebral hemorrhage is associated with poor outcomes or death only in those with stress hyperglycemia, meaning in those who have high sugar levels, but are not diabetic. Dr. Negar Asdaghi:                        Number two, there seems to be an important interaction between the acute glycemic status of the patients and how intensive blood pressure control can prevent hematoma expansion, in that intensive BP control is only effective in prevention of hematoma expansion if the sugar levels are normal. So, a lot of thought-provoking and hypothesis-generating findings, and definitely more to come on this topic. Dr. Negar Asdaghi:                        Moyamoya disease, or MMD, is an idiopathic disorder characterized by progressive stenosis of the supraclinoid internal carotid artery and its main branches in subsequent formation of a network of abnormal lenticulostriate collaterals. First described in Japan, the term "Moyamoya" is a Japanese expression for the puff of smoke and describes the characteristic appearance of the tangled and abnormal collateral vessels that are seen in angiography in various stages of the Moyamoya disease. Dr. Negar Asdaghi:                        Epidemiological studies have revealed several risk factors associated with Moyamoya disease, including Asian ethnicity, female gender, and a family history of the disorder. Given that 15% of MMD patients have a family history of this disease, it's not surprising that genetic factors are suspected to underlie its pathogenesis. Now, a polymorphism in the ring finger protein 213, or RNF213, gene on chromosome 17 has been identified as the strongest genetic susceptibility factor for Moyamoya disease specifically in the East Asian population. Dr. Negar Asdaghi:                        But despite the many advances in understanding the pathophysiology of MMD, as well as advances in animal models and genetic studies, to date, none of the animal models of RNF213 have quite replicated the vascular abnormalities that are typically seen in human Moyamoya disease. Dr. Negar Asdaghi:                        The scientists feel that this is related to how little is known about the exact biological function of RNF213 gene and the protein it encodes. So, in the current issue of the journal, in the study titled "Moyamoya Disease Susceptibility Gene RNF213 Regulates Endothelial Barrier Function," Dr. François Gros-Louis from CHU de Québec Research Center at Laval University in Québec and colleagues aim to study the biological functions of RNF213 using a novel genome editing technology by the name of CRISPR-Cas9 technology. Dr. Negar Asdaghi:                        Joining me now is Dr. Gros-Louis himself to discuss the findings of this paper. Dr. Gros-Louis is a Professor of Neurosciences at the Department of Surgery at Laval University. He holds the Canada Research Chair in Brain Disease Modeling and is the Director of the Induced Pluripotent Stem Cell Platform research in Québec. Dr. Negar Asdaghi:                        Good morning, François. Welcome to our podcast. And thank you so much for joining us. Dr. François Gros-Louis:               My pleasure. Dr. Negar Asdaghi:                        François, you have to promise to hold my clinician's hand through this interview as obviously these are some foreign subjects for us, but very excited to learn from your study and learn from you on the association between RNF213 and the pathophysiology of what happens in Moyamoya disease. Now, before we talk about your paper, can you please talk to us about some basic concepts? What is the RNF213 protein? Dr. François Gros-Louis:               Yes. The RNF213 gene is thought to be involved in mediating protein, protein interactions. The protein also contains a domain which is associated with an ATPase activity. This gene is a susceptibility gene for Moyamoya disease, as you mentioned in the introduction, vascular disorder of intracranial arteries. It's encoded in ubiquitously expressed protein. The protein is found to be expressed throughout the cytocell with the partial association in the intracellular membrane and cytoskeleton. Its expression varies according to the tested tissue type or location or cellular types. Dr. François Gros-Louis:               Although the function of RNF213 protein is unknown, studies suggest that it plays a role in the proper development of blood vessels, cell proliferation, and inflammation. Recently, RNF213 has been reported to be associated with angiogenesis. However, little is known about its endogenous function or its pathogenic role in Moyamoya disease. Our results are in line with these results and indicate that RNF213 could also be a key regulator of cerebral endothelial integrity, whose disruption could be an early pathological mechanism leading to Moyamoya disease. Dr. Negar Asdaghi:                        So, just to continue on this, there's quite a bit of research already done on association of the RNF213 gene, that's located, as we noted earlier, on chromosome 17, and basically susceptibility of development of Moyamoya disease. Can you give our listeners a brief overview of this genetic connections and what was known from past research? Dr. François Gros-Louis:               Yeah, there is a couple polymorphism describing this gene, the most frequent, the most prevalent genetic study have identified the variant R4810K, meaning for arginine is replaced by another amino acid at the position of 4810 within the protein. It's a large protein and a large gene and a susceptible gene and a risk factor for developing Moyamoya disease. Dr. François Gros-Louis:               So, people bearing this variant have a higher chance to develop the disease. This is a loss of function variant, also called inactivating mutation, meaning that the mutated gene product have less or no function. So, this variant is found in heterozygous, meaning one copy, or two copy homozygous in Moyamoya disease patients. While patient bearing homozygous mutation develop a more severe disease with earlier age of onset and worse prognosis, patients bearing heterozygous mutation can also develop the disease. Dr. François Gros-Louis:               So, strong evidence suggests that the carrying rate of RNF213 R4810K mutant is closely related and give a higher chance to develop the disease. Interestingly, also with colleagues, we found that there are other variants within this genes leading to what we think is a gain of function mutation have been associated also with other cerebrovascular disease, such as intracranial aneurysms. Dr. Negar Asdaghi:                        So, François, this is very interesting. Let me recap what you mentioned so I know that I understood it. So, this is an interesting gene, this RNF213, and basically evidence shows that mutations in the RNF213, whether it's loss of function or gain of function, both can result in variety of cerebrovascular disorders. And interestingly, the phenotype of the disease when it comes to loss of function of this gene is actually correlated with whether a person is a carrier, homozygous carrier of this gene, loss of function, or heterozygous carrier of the gene. Dr. Negar Asdaghi:                        So, very interesting information for clinicians who treat patients with Moyamoya disease, specifically those who have a family history of Moyamoya disease, so perhaps a higher chance of carrying a genetic susceptibility gene. Now, we want to get to the paper that you published in this issue of the journal, but I think before we talk about your paper, we also have to have a basic understanding of this CRISPR-Cas9 technology, which is the new genome editing technology that you use in your experiments. Can you please give us a little bit of an overview of this technology? Dr. François Gros-Louis:               Yes. CRISPR-Cas9 gene editing is genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. This technology allows genetic material to be added, removed, or altered at particular location in the genome. Several approaches to genome editing have been developed. Recent one is known as CRISPR-Cas9. So, the CRISPR-Cas9 system has generated a lot of excitement in the scientific community because it is faster, cheaper, and more accurate, and also more efficient than other existing genome editing methods. It's clearly revolutionizing the field in research. Dr. Negar Asdaghi:                        So, it's very exciting. It's truly a new chapter in gene targeting research and editing research. So, now we're ready to hear about your study. And I guess the first part of the study was just to look at how various cells in vitro that you used had expressed RNF213. Can you please tell us about the first part of your experiments? Dr. François Gros-Louis:               Yeah. We first wanted to know where the protein is expressed or where the protein is more highly expressed. So, we found by doing immunofluorescence analysis that the RNF213, so we confirmed that it's ubiquitously expressed in the cytoplasm of different cellular types. So, we found that significant difference also in the expression of RNF213 protein levels in several endothelial cells, where we found it's been highly expressed when compared to other endothelial cells isolated from different other body location, meaning outside of the CNS. So, it's highly expressed also when compared to smooth muscle cells or fibroblasts. Dr. Negar Asdaghi:                        Okay. So, just again, to recap for our listeners, this is, this RNF213 protein, is ubiquitously expressed in many different cell types, but you did find a significantly higher expression rates in endothelial cells, specifically those endothelial cells that were derived from cerebrovasculature. So, that's the first exciting part of the experiments that you showed in the study. Now, using the CRISPR-Cas9 technology, you and your team were able to successfully create an in vitro RNF213 knockout model. Can you please tell us about these models and also the main findings of your study? Dr. François Gros-Louis:               Yeah, so taken together, the results we presented in the article indicate that RNF213 could be a key regulator of cerebral, endothelial and tight junctions integrity whose disruption could be an early pathological mechanisms leading to Moyamoya disease. So, we established for the first time an easily reproducible and stable in vitro 3D model generated using the CRISPR-Cas9 gene editing technology. Dr. François Gros-Louis:               This advanced 3D culture approach has emerged as an excellent system to recapitulate histopathological feature reminiscent to disease pathogenesis. So, 3D cell culture approach is different from standard 2D culture, where cells are cultured, monolayered into a Petri dish. And we have results showing that the 3D cell culture system better mimic the in vivo conditions in terms of cell to cell and cell to matrix interaction and lead to histopathological phenotypic feature can be observed in cell culture, in a 3D fashion. Quite interestingly, alongside of providing the first evidence for the role of RNF213, the maintenance of endothelial barrier and the potential implication of this gene in the expression of maturation of tight junctions. So, we define a novel role for PECAM-1 as well in barrier impairment as a part of the disease pathogenic mechanisms. Dr. Negar Asdaghi:                        Okay. And now this is really interesting. So, I wanted to, again, recap some of the important points that you raised here. First of all, your in vitro models are different than the classic in vitro models, where 2D cells were basically grown in a Petri dish. You are trying to, more and more, replicating what happens, for instance, in blood vessels, where you have endothelial cells overlying mesenchymal cells underneath them, so tunica intima and then tunica media, and so you have 3D cells, where various types of cells are overlying each other in a more in vivo representation of what happens in blood vessels, which is truly interesting. Dr. Negar Asdaghi:                        And what you found was, in sort of summary, was that these knockout endothelial cells ended up having abnormal tight junctions and abnormal connectivity, which basically would lead in an in vivo model to abnormal leaky blood brain barrier, if this were truly in the in vivo model. Does that summarize the findings of the paper? Dr. François Gros-Louis:               Yes, perfectly. Dr. Negar Asdaghi:                        Perfect. And so I want to also give us a chance to talk about the important pro-inflammatory aspects of these knockout cells. You did find that a number of cytokines were expressed in excess in those RNF213 deficient cells. Can you please elaborate on those findings? Dr. François Gros-Louis:               So, to further investigate whether inflammation plays an important role in RNF213-associated Moyamoya disease development, we indeed performed experiments to study pro-inflammatory cytokines and analyze the immune secretome profiles of cerebral RNF213 deficient endothelial cells. So, then the cells can secrete different cytokines or different other proteins. So, by analyzing the secretome, we found an end secretion of a few pro-inflammatory cytokines indicating that inflammation may also play a central role in the initiation of the immune response in the pathogenesis of the disease. Dr. Negar Asdaghi:                        So, this is exciting, François. For years, we thought about the pathophysiology of Moyamoya disease as a disorder involving large vessels. And perhaps the initial thought was that it starts with excessive proliferation of smooth muscles within the middle layer of the cerebral blood vessels, in tunica media, and then perhaps subsequently there will be other abnormalities, including the intimal hyperplasia that is classically seen in Moyamoya. Dr. Negar Asdaghi:                        Your study seems to propose a shift in that pathophysiological paradigm, where the problem seems to start from endothelial cells, so inside of the blood vessels and the tunica intima, and then gradually would go out to the middle layers, and, of course, proposes the hyperinflammatory state in the Moyamoya disease as well. So truly interesting. Do you think that that is the new or rather a paradigm shift for pathophysiology of MMD? Dr. François Gros-Louis:               That's a great question. Our results certainly demonstrated that endothelial cells are involving in the disease pathogenesis in Moyamoya disease, but it doesn't exclude the possibility that other cell types might also be involved in the disease pathogenesis. We know, like you mentioned, that a blood vessel is formed by two different cell layers, tunica intima, media, and adventitia, containing, respectively, endothelial cells, smooth muscle cells, and fibroblasts. So which cells are to be blamed in Moyamoya disease is a question of many ongoing results studies over the years. Dr. François Gros-Louis:               So, using tissue-engineered approach to reconstruct small caliber blood vessels, as we developed in my lab, in combination with patient-derived stem cells, in which adult cells isolated from a patient of any individuals can be reprogrammed into stem cells and re-differentiated into different cell types in occurrence, smooth muscle, fibroblasts, or endothelial cells. We would like to generate blood vessels in which each of the different cellular layers will harbor or not, or a combination with RNF213 mutants. So, this will hopefully help us to elucidate this question. Dr. Negar Asdaghi:                        That's perfect. So, François, before we end the interview, I wanted to ask two more questions. So, what should be our top two takeaway messages from your study? Dr. François Gros-Louis:               We believe that the innovative transdisciplinary approach to generate, for the first time, as we describe in the article, an in vitro 3D model recapitulating important diseases features. So, this model could become a unique tool in precision medicine to study Moyamoya disease or other RNF213-associated pathologies. So, our study provides, for the first time, role of RNF213 in the maintenance of blood-brain barrier and the potential implication of RNF213 in the expression and maturation of tight junctions. Taken together, our data define a novel role for PECAM-1 in the blood-brain barrier impairment in Moyamoya disease. Dr. François Gros-Louis:               So, better characterization of each, also this regulated inflammatory molecules, we found taken separately could reveal a crucial information and help elaborate a more precise approach. Hence, this pro-inflammatory signature could be used as a circulatory biomarker for the follow-up of Moyamoya disease patients and to manage an appropriate treatment, according to the pathology progression. Dr. Negar Asdaghi:                        François, this is great. And last, I want to digress a little bit and ask you about the future of gene editing. I think it's important to end our interview with a little bit of a discussion regarding the future of CRISPR-Cas9 technology. In subatomic quantum physics, people talk about the God particles. And I feel that the CRISPR-Cas9 technology is, in a way, like playing God, if you agree. What do you think is the future for gene editing, and how do you see that helping us in terms of treatment of genetic causes of cerebrovascular disorders? Dr. François Gros-Louis:               Yes, gene editing is, like I said, revolutionizing, of course, experimental therapies for genetic disorder and generated excitement for new and improved gene therapies. We can think that it will be possible to correct any gene mutations associated with a disease to reestablish the normal or natural gene function and help treating the targeted diseases. But also, to me, the future of genome editing also resides in optimizing next generation disease models. The use of genome editing, in particular, the CRISPR-Cas9 technology, has extended to potential in generating new personalized model for a number of disorder, not only including Moyamoya disease or other cerebrovascular diseases, but also diseases like Alzheimer's, ALS, or Parkinson's disease, for which obtaining patient sample is difficult. Dr. François Gros-Louis:               No one wants to give up a bit of his brain. So modeling it, this disease, in vitro will be really helpful in combination also gene editing with the stem cells, induced pluripotent stem cells technology, will allow the generation of better model to mimic human disease and reflects the genetic drivers that govern specific pathology. So, the synergy between IPS cell-based model system and gene editing will play a pivotal role in the root of precision medicine and clinical translation in the future. Dr. Negar Asdaghi:                        Dr. François Gros-Louis, it was a pleasure learning from you. And we look forward to the endless possibilities brought by the future of genome editing technology. Dr. François Gros-Louis:               It was a pleasure discussing with you. Dr. Negar Asdaghi:                        Thank you for joining us. Dr. Negar Asdaghi:                        And this concludes our podcast for the April 2022 issue of Stroke. Please be sure to check out this month's table of contents for the full list of publications, including a series of Focused Updates on the topic of blood pressure management in stroke, organized by Dr. Else Sandset. I would also like to draw your attention to two scientific statements from the American Heart Association, which appear in print in the April issue. The first one is titled "Identifying Best Practices to Improve Evaluation and Management of In-Hospital Stroke," and the second one is on the effect of marijuana use on brain health. Dr. Negar Asdaghi:                        And now, to end our podcast, last month, in honor of the 2022 Olympic Games, and to celebrate those with determination to survive and push despite the most difficult of circumstances, we ended our podcast with the story of a refugee Olympic athlete. Dr. Negar Asdaghi:                        Sadly, since our last podcast, the world has seen even darker days of war, mass immigration, displacement, and human suffering. At times like this, we're reminded that although not all of us can help everyone, but at least each of us can do something to help someone, and the comfort in knowing that what we do in the field of medicine, from daily patient care to the scientific work leading to the next medical breakthrough, every action is a step forward in reducing the suffering of another person. And what better way to do this than staying alert with Stroke Alert. Dr. Negar Asdaghi:         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, visit AHAjournals.org.

Leitura bíblica 1 Livra-me, ó Deus, dos meus inimigos;livra-me daqueles que se levantam contra mim!2 Livra-me desta gente que pratica a iniquidade e o crime!3 Armam ciladas contra a minha vida;preparam-se para me cair em cima,sem que eu lhes tenha feito mal algum, ó Senhor.4 Agitam-se e têm pressa em me liquidar, sem razão.Desperta e ajuda-me!5 Desperta para me ajudares, Senhor,Deus dos exércitos, Deus de Israel!Castiga as nações pecadoras que nos cercam;não poupes nenhum desses maus e traiçoeiros!6 Ao anoitecer vêm espiar-me,andando em volta como cães, rondando a cidade.7 Ouço-lhes os insultos que ferem como espadas.Gritam e dizem: “Ninguém nos ouve!”8 Mas tu, Senhor, ris-te deles;também vês como essas nações são ridículas.9 Ó Deus, tu és a minha força!Espero em ti porque és a minha defesa segura.10 Deus nunca mudará o seu amor por mim;fará com que veja cumprido o meu desejo,a respeito dos meus inimigos.11 Não os mates, pois o meu povo logo se esquece.Que o teu poder os disperse e abata, Senhor,pois és o nosso escudo!12 Pecam ao falar! São uns arrogantes!Só proferem maldições e mentiras.Por essas mesmas coisas serão condenados.13 Destrói-os com a tua severidade e liquida-os,para que se saiba que Deus governa em Jacobe que domina sobre toda a Terra!14 Ao anoitecer vêm espiar-me,andando em volta como cães, rondando a cidade.15 Uivam e procuram comida,para matar a fome.16 Quanto a mim, não deixarei de cantar a tua força;cedo pela manhã cantarei, com alegria, o teu amor.Pois tens sido o meu refúgio bem seguro,a minha segurança nos momentos de angústia.17 A ti, pois, minha força, cantarei louvores;tu és o Deus que me defende e me ama!Salmo 59 (O Livro) Devocional Há ciclos na nossa vida em que as coisas parecem ir de mal a pior. Pomos a cabeça fora da toca e só vemos lobos a cirandar de um lado para o outro, avançando determinados para nos abocanhar a alma. Há gente com sede de fazer sangue e logo à nossa custa. É exactamente nesses momentos que importa pedir a Deus que repare na nossa aflição. Não que Ele Se distraia por um instante que seja, acontece que nós é que precisamos de nos relembrar que temos a Sua ajuda à mão de semear. É que enquanto Lhe apresentamos os detalhes, que aliás são sempre do Seu inteiro conhecimento, vamos desabafando e sossegando. Sobretudo quando Lhe confidenciamos entre dentes: “Desperta e vem em meu auxílio!” Sim, “é Ele o nosso protector” diante de rosnadelas e flechadas. Até podemos ser alvo de chacota, mas no meio de tanta ameaça e zombaria é Deus Quem se ri por último. E nós com Ele, pois debaixo de fogo, podemos dar-nos por seguros: “Deus que nos ama, virá ao nosso encontro” e nos dará a vitória. Atenção, porém, que o triunfo será à Sua maneira e não à nossa. Incorremos no erro de reivindicar juízo implacável para terceiros, ansiando pelo exercício da Sua compaixão quando somos nós a estar na berlinda. Não esqueçamos que a justiça que requeremos para os outros é igual para nós. Desabafemos com Ele, mas deixemos também nas Suas mãos a resolução da injustiça alheia. Quanto à que nos assiste, cantemos o Seu poder, o Seu amor e a Sua graça a cada manhã, pois é Ele mesmo, continuamente, a nossa escapatória

Pecam os militantes do ateísmo que propagam mentiras e todos aqueles que blasfemam contra o Eterno Deus.

A graça é a verdade que liberta você, e não cumprir os mandamentos, simplesmente porque você não consegue! Quanto mais você estiver sob a lei, mais o pecado ganha força.

Tatt opp i Lunkent verksted 12 juni, 2018Kunst-sosiolog, seniorforsker for IRIS og Anna Ihles andre ønskegjest: Merete Jonvik besøker "Verkstedet" en lunken sommerkveld og vi snakker om at hun har kuttet ut sitt mellomnavn, LUNKENT BEKJENTSKAP hennes noe uheldige minne, å gå på kunst-ting i Stavanger, hennes forhold til Stavanger, hennes tidligere bosteder, hennes barns forhold til Stavanger, mitt forhold til Stavanger, hvorfor hun flyttet til Stavanger, hennes doktorgrad, når og hvor hun starter å gå på kunst-åpninger, nysgjerrigheten hennes rundt salget av Barbara Hepworth skulpturen, lunken skål, Meretes forhold til kaffi-drikking, min fars kaffi vaner, hennes forhold til Tove Kommedal, Hans Edward Hammonds og Anna Ihle, Arnhild Sunnanå, Prosjektrom Normanns, mine komfortsoner i kunstsammenheng, mine tryggheter og utryggheter i Kunståpning settinger, Margrethe Aanestad, Bruksrommet, Ingrid Toogood, 10 års arbeidsstipend for kunstnere, Meretes lille tekst i norsk kunstårbok 2018, å skrive på nynorsk, BERGMAN, LUNKEN DAGBOK, å utforske sitt eget sinn, når Merete tok personlighetstesten Big 5, for lite og for mye selvinnsikt, SELVSKRYT, bilister, syklister, bussister og passasjerister, min selvskryt, A mennesker og B mennesker, Meretes penn, jeg forteller om en Lunken Kaffi høydare, Ka då ittepå 2018 og så snakker vi litt om Stavangers kunstmiljø idag før vi avslutter kjapt og presist.God lytting! View fullsize

Chemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments, a better understanding of biochemical changes in the resistant tumors is needed. Therefore, especially in vivo models are very important tools to generate standardized cell-material, which can be examined by high throughput techniques. Thus, it should be possible to find new targets for therapy or even for diagnostic. This thesis focusses on the characterization of the in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft mouse model. First step of the work was the establishment of the xenograft mouse model. SCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. After 10 weeks of response to the therapy, the tumor growth relapsed and tissue grew with very fast doubling time again, despite of ongoing treatment. This aggressive manner of growth under therapy could be also observed in a re-implantation study where the reisolated CPA chemoresistant HUH-REISO tumors grew without a lag phase, indicating an endogenous imprinted component. To evaluate this, tumors were examined by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Histochemical analysis of HUH-REISO tumors revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO in comparison to parental HUH-7 cells and in vivo passaged HUH-PAS cells (in vivo grown without chemotherapeutic CPA pressure). The pronounced network of host murine vascularization in parental HUH-7 tumors was completely substituted by a network of human and murine vessel-like structures in HUH-REISO tumors under therapy. In addition, cell lines of these tumors were analyzed in endothelial trans-differentiation studies on matrigel. In those studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. Such a trans-differentiation capacity requires a lineage of cells with pluripotent capacities like so called tumor stem cells. Indeed, I could show in a comparative study on stemness and plasticity markers that Thy-1, Oct-4, Sox-2 and Nanog were upregulated in resistant xenografts. Furthermore, under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression, which I could also show by qRT-PCR. Notch-1 raised in HUH-PAS under therapeutic pressure, meanwhile it was conversely regulated in comparison to Thy-1, Oct-4, Sox-2 and Nanog in HUH-REISO. In both groups Notch-3 was inducible by 2 times CPA treatment and fell back on base level after further four therapeutic cycles in HUH-REISO. To conclude all these finding: chemoresistance of HUH-REISO was not manifested under standard in vitro, but only under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of trans-differentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.

Background: Chemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments a better understanding of biochemical changes in the resistant tumors is needed. In this study, we focus on the characterization of in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft model. Methods: SCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. Tumors were evaluated by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Cell lines of these tumors were analyzed by qRT-PCR and in endothelial transdifferentiation studies on matrigel. Results: HUH-REISO cells, although slightly more sensitive against activated CPA in vitro than parental HUH-7 cells, fully retained their in vivo CPA chemoresistance upon xenografting into SCID mice. Histochemical analysis of HUH-REISO tumors in comparison to parental HUH-7 cells and passaged HUH-PAS cells (in vivo passaged without chemotherapeutic pressure) revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO. In transdifferentiation studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. A comparative study on stemness and plasticity markers revealed upregulation of Thy-1, Oct-4, Sox-2 and Nanog in resistant xenografts. Under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression. Conclusions: Chemoresistance of HUH-REISO was not manifested under standard in vitro but under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of transdifferentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.

We assessed whether Angiopoietin-2 (Ang2), a Tie2 ligand and partial antagonist of Angiopoietin-1 (Ang1), is required for early vessel destabilization during postischemic angiogenesis, when combined with vascular growth factors. In vitro, matrigel co-cultures assessed endothelial-cell tube formation and pericyte recruitment after stimulation of VEGF-A, Apelin (APLN), Ang1 with or without Ang2. In a murine hindlimb ischemia model, adeno-associated virus (rAAV, 3×10(12) virusparticles) transduction of VEGF-A, APLN and Ang1 with or without Ang2 (continuous or early expression d0-3) was performed intramuscularly (d-14). Femoral artery ligation was performed at d0, followed by laser doppler perfusion meassurements (LDI) 7 and 14. At d7 (early timepoint) and d14 (late timepoint), histological analysis of capillary/muscle fiber ratio (CMF-R, PECAM-1) and pericyte/capillary ratio (PC-R, NG2) was performed. In vitro, VEGF-A, APLN and Ang1 induced ring formation, but only APLN and Ang1 recruited pericytes. Ang2 did not affect tube formation by APLN, but reduced pericyte recruitment after APLN or Ang1 overexpression. In vivo, rAAV.VEGF-A did not alter LDI-perfusion at d14, consistent with an impaired PC-R despite a rise in CMF-R. rAAV.APLN improved perfusion at d14, with or without continuous Ang2, increasing CMF-R and PC-R. rAAV.Ang1 improved perfusion at d14, when combined with rAAV.Ang2 (d0-3), accompanied by an increased CMF-R and PC-R. The combination of early vessel destabilization (Ang2 d0-3) and continuous Ang1 overexpression improves hindlimb perfusion, pointing to the importance of early vessel destabilization and subsequent vessel maturation for enhanced therapeutic neovascularization.

Chronische Extremitätenischämien stellen eine sowohl subjektiv belastende als auch volkswirtschaftlich bedeutende Krankheitsentität dar. Dabei können etliche Patienten mit den zur Verfügung stehenden konventionellen Verfahren nicht befriedigend therapiert werden. Neuere Konzepte zur Zelltherapie der chronischen Therapie führten in der klinischen Erprobung dabei zu eher ausbaufähigen Resultaten. Unsere Gruppe konnte in Vorarbeiten zeigen, dass die exogene Applikation embryonaler Endothelprogenitorzellen (eEPCs) im Tiermodell zu einem deutlichen Effet auf die chronische Ischämie führt. Um diesen Effekt weiter zu steigern, wurde ein künstliches Fusionsmolekül aus dem Chemokin SDF-1 als Kopf, der Mucindomäne des Fractalkine als Rückgrat und einem GPI-Teil zur Verankerung im Endothel (SDF-Fractalkine-GPI oder S1FG) kloniert. Wir konnten ebenfalls in Vorarbeiten zeigen, dass dieses S1FG eEPCs in vitro und in vivo rekrutiert, und dass eine Vortransfektion des Endothels des Ischämiegebietes vor Applikation der eEPCs zu einer Steigerung des funktionellen Effekts führt. Wir stellten die Hypothese auf, dass ein Ersatz der exogenen Applikation der eEPCs durch eine Mobilisierung endogener vaskulärer Progenitorzellen ebenfalls zu einem guten funktionellen Effekt führt. Weiterhin sollte der genaue Rekrutierungsmechanismus des S1FG untersucht werden. Zur Untersuchung des funktionellen Effekts wurde wie in den Vorarbeiten ein Kaninchenmodell der chronischen Hinterlaufischämie gewählt, bei dem an Tag 0 die rechte Femoralarterie entfernt wurde. Nach Entwicklung eines chronischen Zustandes wurde am Tag 7 eine Angiographie beider Femoralisstromgebiete durchgeführt und entweder S1FG oder eGFP liposomal per Retroinfusion transfiziert. An den Tagen 9, 10 und 11 wurde jeweils 1 mg des kurzwirksamen CXCR4-Antagonisten AMD3100 oder 1 ml NaCl intraperitoneal injiziert. An Tag 35 wurde eine erneute Angiographie durchgeführt, das Tier getötet und die Hinterlaufmuskulatur entnommen. Die Angiogenese wurde über die mittels PECAM-1-Färbung bestimmte Kapillardichte gemessen, die Arteriogenese über die Mengenzunahme der in der Angiografie sichtbaren Kollateralen. Zur Messung der Perfusion wurden die Flussgeschwindigkeit in der Angiographie sowie fluoreszierende Mikrosphären verwendet. Um das Rekrutierungsprofil des S1FG in vitro zu eruieren, wurden statische Adhäsionsversuche mit PMNs auf transfizierten HMECs sowie Adhäsionsversuche in Flusskammern von THP-1-Zellen auf transfizierten HUVECs verwendet. Es zeigte sich, dass signifikant weniger PMNs auf S1FG-transfiziertem Endothel adhärieren, als auf Fractalkine-transfizierten HMECs, während die eEPC-Adhäsion signifikant besser war. Bei den Versuchen unter Schubspannung ergab sich durch S1FG-Transfektion keine signifikante Änderung der Anzahl rollender Zellen, während die Anzahl fest haftender Zellen signifikant höher war. Durch Zugabe eines L-Selektin-Antikörpers zu den THP-1-Zellen vor Superfusion konnte die Anzahl fest haftender Zellen wieder auf Kontrollniveau reduziert werden, während die Anzahl rollender Zellen leicht reduziert wurde. Zugabe von AMD3100 führte dort nicht zu einer signifikanten Änderung der Anzahl adhärierender Zellen, jedoch wurde durch AMD3100 die Stärke der Interaktion, gemessen als Anzahl nach Applikation hoher Flüsse noch adhärierender Zellen, wieder auf Kontrollniveau reduziert. Ein über andere Adhäsionsmoleüle vermitteltes Zellrollen ist also vermutlich eine Voraussetzung für eine adäquate S1FG-Funktion. Dabei würde es über SDF-1-CXCR4-Interaktion zu einer Erhöhung der Festigkeit der Bindung kommen. In Bezug auf den funktionellen Effekt im Tiermodell führte Verwendung von S1FG und AMD3100 zu einer signifikanten Steigerung von Kapillardichte, Kollateralenwachstum und Perfusion gegenüber der Kontrolle. Die Werte lagen dabei im selben Bereich wie durch Verwendung von S1FG und eEPCs erzielte Ergebnisse. Transfektion von S1FG ohne weitere Behandlung führte nicht zu einer signifikanten Änderung eines Parameters zur Kontrolle, während die Verwendung von AMD3100 zu moderaten Steigerungen bei Kapillardichte, und Kollateralenwachstum führte. Die Werte waren jedoch immer noch signifikant geringer als die nach Kombination von S1FG und AMD3100 erreichten Werte. Verwendung einer proteaseresistenten, funktionell jedoch aktiven Mutante für das SDF-1 im S1FG-Molekül führte nicht zu signifikanten Änderungen bei funktionellen Parametern, bis auf eine zwar signifikante, quantitativ jedoch geringe Verringerung des Kollateralwachstums. Zusammenfassend kann man sagen, dass die lokale Applikation eines künstlichen Adhäsionsmoleküls gemeinsam mit einer Mobilisierung knochenmarksständiger endothelialer oder vaskulärer Progenitorzellen zu einem deutlichen funktionellen Effekt führt, der den der Zellmobilisierung ohne Adhäsionssteigerung übertrifft. Dennoch birgt der Ansatz einige Risiken, wie die versehentliche Förderung von Tumorangiogenese oder die Beschleunigung des Wachstums atherosklerotischer Plaques. Zusätzlich bleibt unklar, welche Zellen genau durch das Adhäsionsmolekül rekrutiert werden, und ob es sich überhaupt um eine homogene Zellpopulation handelt. Weiterhin bleibt zu überprüfen, in welcher Weise die Wirksamkeit der Therapie durch chronische Defekte der Progenitorzellmobilisierung und -funktion, wie sie beispielsweise bei Diabetes oder Nikotinabusus auftreten, beeinträchtigt wird, und ob gegebenenfalls Optimierungsmöglichkeiten in Bezug auf das Mobilisierungsregime, den Aufbau des Adhäsionsmoleküls oder die Applikationsart bestehen. Nichtsdestotrotz stellt diese Methode einen vielversprechenden neuen Ansatz zur Verbesserung der bisher eher zwiespältigen Ergebnisse der Zelltherapie dar.

Background/Aims: Reduction of capillary network density occurs early in the development of metabolic syndrome and may be relevant for the precipitation of diabetes. Agonists of the peroxisome proliferator-activated receptor (PPAR)-gamma transcription factor are vasculoprotective, but their capacity for structural preservation of the microcirculation is unclear. Methods: Male Wistar rats were rendered diabetic by streptozotocin and treated with pioglitazone in chow for up to 12 weeks. Capillary density was determined in heart and skeletal muscle after platelet endothelial cell adhesion molecule-1 (PECAM-1) immunostaining. Hallmarks of apoptosis and angiogenesis were determined. Results: Capillary density deteriorated progressively in the presence of hyperglycemia (from 971/mm(2) to 475/mm(2) in quadriceps muscle during 13 weeks). Pioglitazone did not influence plasma glucose, left ventricular weight, or body weight but nearly doubled absolute and relative capillary densities compared to untreated controls (1.2 vs. 0.6 capillaries/myocyte in heart and 1.5 vs. 0.9 capillaries/myocyte in quadriceps muscle) after 13 weeks of diabetes. No antiapoptotic or angiogenic influence of pioglitazone was detected while a reduced expression of hypoxia-inducible factor-3 alpha and PPAR coactivator-1 alpha (PGC-1 alpha) mRNA as well as vascular endothelial growth factor (VEGF) protein possibly occurred as a consequence of improved vascularization. Conclusion: Pioglitazone preserves microvascular structure in diabetes independently of improvements in glycemic control and by a mechanism unrelated to VEGF-mediated angiogenesis. Copyright (C) 2012 S. Karger AG, Basel

The emigration of leukocytes from the circulation is a critical step during immune surveillance and inflammatory reactions that is governed by a coordinated interplay involving a spectrum of adhesion and signal molecules. While a great deal has been learned about the early steps of leukocyte recruitment, i.e. rolling and adhesion, little is known about the subsequent steps, transendothelial and interstitial migration when leukocytes migrate across the endothelial layer lining the blood vessel and move to the sites of inflammation. In particular, it is not fully understood which endothelial receptors are responsible for extravasation of leukocytes into the perivascular space. Moreover, the mechanisms of interstitial migration of leukocytes during inflammation remain to be clarified in vivo. In the first part of the study, we analyzed the role of ESAM for leukocyte migration in vivo. ESAM is a novel adhesion receptor which is specifically expressed at endothelial tight junctions and on platelets. Using RLOT intravital microscopy of the murine cremaster muscle, we have shown that IL-1β-induced leukocyte transmigration was reduced by about 50% in ESAM-deficient mice without affecting leukocyte rolling and adhesion. In summary, ESAM at endothelial tight junctions participates in the migration of neutrophils through the vessel wall. In the second part of the study, we investigated the role of two other recently discovered receptors, CD99 and CD99L2, for leukocyte migration. Similar to ESAM, these receptors are expressed at endothelial cell contacts but did not belong to any of the known protein families. We demonstrate that CD99 and CD99L2 mediate transendothelial migration of neutrophils in vivo without any effect on leukocyte rolling and adhesion. Finally, we show that the inhibitory effect of anti-CD99 and CD99L2 antibodies on cytokine-induced leukocyte transmigration in cremasteric venules is amplified in PECAM-1-/- mice. This fact suggests that a functional relationship between PECAM-1 and CD99/ CD99L2 might exist in mediating leukocyte transmigration. Taken together, our study provides the first evidence for a role of CD99 and CD99L2 in the process of leukocyte transendothelial migration in vivo. In the third part of the study, we established a novel approach allowing the visualization and analysis of directional leukocyte interstitial migration in vivo. Our technique combines RLOT and multicolor fluorescence microscopy with microinjection for local application of chemoattractants. In the mouse cremaster muscle, we show that microinjection of chemoattractants (MIP-1α and PAF) induced directional leukocyte polarization and migration. Combination of RLOT microscopy with fluorescence microscopy allowed simultaneous visualization and analysis of migratory behavior of different leukocyte subsets upon chemotactic stimulation. Moreover, this approach enabled an imaging of subcellular events such as mitochondria redistribution in single polarized interstitially migrating leukocytes in vivo. This technique opens new avenues for investigations of the mechanisms of interstitial migration of leukocytes as well as the observation of morphological changes and subcellular events in different leukocyte subsets during their interstitial migration in vivo.

Thu, 13 Nov 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/9332/ https://edoc.ub.uni-muenchen.de/9332/1/Huff_Ines.pdf Huff, In

Wed, 2 Jul 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/8943/ https://edoc.ub.uni-muenchen.de/8943/1/Lamparter_Mathias.pdf Lamparter, Mathias ddc:500, ddc:570, Fakultät für Bio

Die vorliegende Arbeit beschäftigt sich mit der funktionellen und molekularen Charakterisierung von humanen CD34- Zelllinien aus dem peripheren Blut (V54/1, V54/2) im Vergleich zu den aus dem Knochenmark etablierten Zelllinien (L87/4, L88/5). Die Klone V54/1 und V54/2 wurden aus dem peripheren Blut nach Stammzellmobilisierung und CD6 Depletion durch Zugabe eines Faktorengemisches aus IL-1b, IL-3, IL-6, IL-7, IL-8 und IL-11 erzeugt. L87/4 und L88/5 hingegen sind adhärente und wachstumsarretierte Stromazellen, die die Erhaltung und Differenzierung von hämatopoetischen Vorläuferzellen durch Mediatoren ermöglichen (Thalmeier et al. 2000). Das Ziel dieser Arbeit war die Untersuchung von Stammzelleigenschaften bei den Zelllinien L87/4, L88/5, V54/1 und V54/2. Dazu soll die Färbung mit den Farbstoffen Rhodamin 123 (Rh123) und Hoechst 33342 zeigen, ob Subpopulationen innerhalb der Klone mit unterschiedlichen Färbeeigenschaften, bestehen. Die biologische Bedeutung der beiden Farbstoffe liegt darin, dass Sie dazu geeignet sind frühe Stammzellen zu identifizieren. Als Substrat der P-Glykoproteinpumpe, die u.a. auf frühen Vorläuferzellen mit stark erhöhter Repopulationskapazität gefunden wird, werden diese Farbstoffe aus der Zelle gepumpt. Der Farbstoff-Efflux kommt durch die mdr-Gen-kodierte (multi-drug-resistance) und Kalzium-abhängige P-Glykoproteinpumpe zustande. Das P-Glykoprotein hat neben der Bedeutung in der Stammzellbiologie in der angewandten Medizin eine wichtige Funktion in der Resistenzentwicklung von Tumoren. Des weiteren wurden bei den Zelllinien stammzellrelevante Oberflächenantigene (CD10, CD34, CD14, CD105, SH3 und CD117) untersucht, um Unterschiede zwischen L87/4, L88/5 und den Klonen V54/1, V54/2 zu erkennen. Versuche zur Induktion der Differenzierung sollten Hinweise auf die Plastizität der Zelllinien geben. Experimente an den durch den Rh123-Efflux unterscheidbaren Subpopulationen der Zelllinie V54/2 dienen der Aufklärung von Unterschieden in Morphe, zellulären Transportfunktionen und Funktionseinheiten von Transkriptionsfaktor Netzwerken. Methodisch wurde für die Analyse der Epitope und der Färbungen mit Rh123 und Hoechst 33342 ein Durchflußzytometer verwendet. Die Analyse der Funktionseinheiten von Transkriptionsfaktor Netzwerken wurde mittels Reverse Transkriptase Polymerase Ketten Reaktion durchgeführt. Die Ergebnisse der Färbeexperimente zeigten, dass bei allen untersuchten Zelllinien durch eine unterschiedliche Anfärbbarkeit der Zellen mit dem Farbstoff Rh123 zwei Subpopulationen unterschieden werden können. Die jeweils größere Subpopulation der Zelllinien färbt sich mit Rh123 an und bleibt auch nach einer definierten Inkubationszeit, die den Rh123-Efflux ermöglichen soll, gefärbt. Sie wird Rh123high genannt. Die übrigen Zellen, die bei allen Zelllinien unter 10% der Gesamtpopulation betragen, sind in der Lage den Farbstoff aus der Zelle zu pumpen. Diese Subpopulation wird Rh123low genannt und ist mit Stammzelleigenschaften wie tausendfach erhöhter Repopulationsfähigkeit in NOD/SCID-Mäusen assoziiert. Es konnte also innerhalb der untersuchten monoklonalen Linien eine Rh123low Subpopulation identifiziert werden, die sich durch zahlreiche biologische Eigenschaften von der Gesamtpopulation unterscheidet. Da der Rh123 Efflux durch eine Kalzium-abhängige Pumpe zustande kommt, lässt sie sich durch den Kalziumantagonisten Verapamil hemmen. Eine Hemmung der Pumpe bewirkt, dass die Rh123low Zellen nicht mehr in der Lage sind Rh123 aus der Zelle zu pumpen, so dass sie nach einer definierten Inkubationszeit mit Rh123 gefärbt bleiben. Neben diesem funktionellen Beweis für die P-Glykoproteinpumpe konnte durch den strukturellen Nachweis der Pumpe mittels eines Antikörpers gegen P-Glykoprotein ein definitiver Beweis für das Vorhandensein der aktiven P-Glykoproteinpumpe bei der Rh123low Population erbracht werden. Mit dem anderen Farbstoff Hoechst 33342 können die jeweiligen Anteile der Zelllinien in den einzelnen Stadien des Zellzyklus nachgewiesen und zudem ein kleiner Anteil an Zellen bestimmt werden, der als „Side Population“ (SP-Zellen) definiert wird. Diesen SP-Zellen werden Eigenschaften von aktiven Stammzellen zugeschrieben. Hierbei besteht ein Unterschied zwischen den aus dem Knochenmark und den aus dem peripheren Blut etablierten Linien, da die Zellen aus dem peripheren Blut nicht nur ein anderes Zellzyklusmuster aufweisen, sondern auch einen höheren Anteil an SP-Zellen besitzen. Es wurden vergleichende Untersuchungen zwischen den Zelllinien und zwischen den Rh123high und Rh123low Subpopulationen innerhalb einer Zelllinie mit Antikörpern gegen die Epitope CD14, CD45, HLA-DR, CD10, CD117, CD105 und SH3 durchgeführt. Dabei waren CD14 und CD45 auf allen Zelllinien negativ, wobei alle Zelllinien eine positive Expression für den mesenchymalen Marker Endoglin (CD105) und für SH3 (CD73) zeigten. CD117 konnte nur auf den aus dem Knochenmark etablierten Zelllinien L87/4 und L88/5 nachgewiesen werden. CD34, ein charakteristischer Marker für hämatopoetische Vorläuferzellen, aber auch für Endothelzellen, konnte nur auf den Zellen der Rh123low Subpopulation nachgewiesen werden. Im Gegensatz dazu exprimieren die Rh123high Zellen kein CD34. Da es sich bei den Zelllinien um Klone handelt, ist der Unterschied in der Expression von CD34 zwischen der Rh123low und der Rh123high Population ein deutlicher Hinweis auf die Plastizität der Zelllinien und das Fließgleichgewicht zwischen Rh123low und Rh123high. Durch eine Zellsortierung der Zelllinie V54/2 wurde die Rh123low von der Rh123high Subpopulation getrennt, um sie dann bezüglich ihrer Morphologie, dem Wachstum in Methylzellulose und der Expression ausgewählter Funktionseinheiten von Transkriptionsfaktor Netzwerken zu untersuchen. Dabei erhärtete sich die Hypothese, dass es sich bei der Rh123low Subpopulation um aktivere Zellen mit einer gesteigerten Expression von erythroid/myeloischen und mesodermalen Eingaben (z.B. VEGF, BMP-4), Rezeptoren (z.B. tie-1), vernetzter Transkriptionsfaktoren (z.B. GATA, ETS) und letztendlich Ausgaben (z.B. PECAM) handelt. Diese fungieren in Netzwerken mit dem Ziel, stammzellrelevante Funktionen zu ermöglichen. Die Morphologie zeigte in den Zytozentrifugationspräparaten deutliche Unterschiede zwischen Zellen der Rh123low und der Rh123high Subpopulation. Die Rh123low Subpopulation besteht aus lymphoid-ähnlichen Zellen, was für Zellen mit Stammzellfunktion charakteristisch ist. Die Rh123high Subpopulation dagegen hat ein insgesamt größeres Zellvolumen und einen gebuchteten Kern mit perinukleärer Aufhellung. Untersuchungen des klonalen Wachstums in der Methylzellulose ergaben bei keiner der Subpopulationen eine wesentliche Koloniebildung. Durch die Inkubation der Zelllinie V54/2 mit dem Neurotropen Wachstumsfaktor (NGF) konnte eine morphologische Änderung in Richtung einer neuronalen/glialen Differenzierung nach 8-12 Stunden induziert werden. Der immunhistochemische Nachweis von Glial Fibrillary Acidic Protein (GFAP) bestätigte die mesenchymale Potenz zumindest in Richtung einer glialen Differenzierung. Das unterschiedliche Expressionsmuster ausgewählter, für die Differenzierung notwendiger Zusammenspieler innerhalb von Transkriptionsfaktor Netzwerken innerhalb der Rh123high und der Rh123low Population bei V54/2 war ein weiterer Hinweis, dass es sich bei der Rh123low Subpopulation um aktive Vorläuferzellen mit möglicher Stammzellpotenz handelt. In der Rh123low Subpopulation wurde im Gegensatz zur Rh123high Population eine Expression von BMP4, GATA1, GATA3 nachgewiesen, die essentiell für die Hämatopoese und für eine mesenchymale Differenzierung ist. Die Faktoren für GATA2, GATA3, beta globin, Elf-1 und PECAM1 wurden in einem stärkeren Maß in der Rh123low als in der Rh123high Population exprimiert. BMP-Rez., Myb, sowie die Endothel-assoziierten Faktoren Tie-1 und VEGF waren in beiden Subpopulationen gleich stark vorhanden. Bei den wenigen Funktionseinheiten der größeren und Rh123high Population handelt es sich vor allem um angiogenetische Faktoren, was auf eine limitierte Differenzierungseigenschaft der Rh123high Subpopulation und die enge Beziehung zwischen Blut- und Endothelzellen („Hämangioblast“) hinweist. Ein Nachweis für die Plastizität der Stammzellen innerhalb der von uns etablierten Zelllinien wurde dadurch erbracht, dass die zellsortierten Subpopulationen Rh123low und Rh123high nach dem Sortierexperiment getrennt rekultiviert wurden, wobei das Wachstum der Rh123low Subpopulation deutlich langsamer war als das der Rh123high Subpopulation. Nach zwei Wochen wurden die zellsortierten Subpopulationen erneut einer Rh123 Färbung unterzogen, wobei sich wiederum das ursprüngliche Verhältnis zwischen den Rh123low und Rh123high Subpopulationen einstellte. So kann man aus der Transdifferenzierung der Zelllinien von Rh123low in Rh123high und umgekehrt die Plastizität der hier untersuchten adulten Stammzelllinien ableiten. Die Ergebnisse sollen zum grundlegenden Verständnis der Biologie adulter (nicht embryonaler) Stammzellen beitragen und damit die Möglichkeit schaffen, adulte Stammzellen bzw. deren Subpopulationen gezielt für einen reparativen Gewebe- und Organersatz zu verwenden. Dabei liefern sie die Basis für weitergehende Untersuchungen zum besseren Verständnis der physiologischen und regenerativen Vorgänge, z.B. auch bei Alterung oder bei gesteigerter Funktion. Darüber hinaus kann aufgrund der vorliegenden Ergebnisse durch weitere Untersuchungen möglicherweise besser verstanden werden, ob es gelingen kann das Potential adulter Stammzellen zur therapeutischen Gewebereparation, z.B. zur Verhinderung oder Verringerung einer Narbenbildung, zu nutzen.

Background: Molecular mechanisms regulating leukocyte sequestration into the tissue during endotoxemia and/or sepsis are still poorly understood. This in vivo study investigates the biological role of murine PECAM-1 and VCAM-1 for leukocyte sequestration into the lung, liver and striated skin muscle. Methods: Male BALB/c mice were injected intravenously with murine PECAM-1 IgG chimera or monoclonal antibody (mAb) to VCAM-1 ( 3 mg/kg body weight); controls received equivalent doses of IgG2a ( n = 6 per group). Fifteen minutes thereafter, 2 mg/kg body weight of Salmonella abortus equi endotoxin was injected intravenously. At 24 h after the endotoxin challenge, lungs, livers and striated muscle of skin were analyzed for their myeloperoxidase activity. To monitor intravital leukocyte-endothelial cell interactions, fluorescence videomicroscopy was performed in the skin fold chamber model of the BALB/c mouse at 3, 8 and 24 h after injection of endotoxin. Results: Myeloperoxidase activity at 24 h after the endotoxin challenge in lungs (12,171 +/- 2,357 mU/g tissue), livers ( 2,204 +/- 238 mU/g) and striated muscle of the skin ( 1,161 +/- 110 mU/g) was significantly reduced in both treatment groups as compared to controls, with strongest attenuation in the PECAM-1 IgG treatment group. Arteriolar leukocyte sticking at 3 h after endotoxin (230 +/- 46 cells x mm(-2)) was significantly reduced in both treatment groups. Leukocyte sticking in postcapillary venules at 8 h after endotoxin ( 343 +/- 69 cells/mm(2)) was found reduced only in the VCAM-1-mAb-treated animals ( 215 +/- 53 cells/mm(2)), while it was enhanced in animals treated with PECAM-1 IgG ( 572 +/- 126 cells/mm(2)). Conclusion: These data show that both PECAM-1 and VCAM-1 are involved in endotoxin-induced leukocyte sequestration in the lung, liver and muscle, presumably through interference with arteriolar and/or venular leukocyte sticking. Copyright (C) 2004 S. Karger AG, Basel.