Abstract 162: Resident Aortic Intimal Mononuclear Phagocytes (MNPs) in the Promotion of Atherosclerosis

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jesse W Williams ◽  
Ki-Wook Kim ◽  
Stoyan Ivanov ◽  
Slava Epelman ◽  
Kory Lavine ◽  
...  
Keyword(s):  
Disease Progression ◽  
Cell Tracking ◽  
Lineage Tracing ◽  
Mononuclear Phagocytes ◽  
Gm Csf ◽  
Reporter Mice ◽  
En Face ◽  
A Cell ◽  
Functional Analyses ◽  
First Time

Atherosclerosis is an underlying cause of cardiovascular disease (CVD) and a leading cause of morbidity and mortality worldwide. Atherosclerosis promotes CVD through plaque formation, restricted blood flow, and thrombotic events. Macrophage accumulation in plaques, their uptake of cholesterol, and subsequent local death drive disease progression, however, there is growing appreciation for more diverse roles of myeloid cells during disease progression. Here, we focused on characterization of so-called vascular dendritic cells (DCs), which we refer to as aortic mononuclear phagocytes (MNP), that reside under the endothelium in plaque-prone areas. Using en face whole-mount confocal microscopy of aortas, we confirm a uniform resident CD11c+ CX3CR1+ MHCII+ MNP population, even in C57/BL6 mice resistant to atherosclerosis. We find aortic MNPs require M-CSF and Flt3 signaling for survival, but are independent of CCR2 and GM-CSF receptor signaling, making them a distinct myeloid population. They express macrophage-restricted genes LysM and CD64, but not dendritic cell specific genes zBTB46 or L-myc. Lineage-tracing analysis using CD115creER and Flt3cre reporter mice indicate that aortic MNPs likely differentiate from definitive hematopoiesis and not early yolk sac progenitors. Parabiosis experiments show that aortic MNPs are self-maintained independent of blood-born progenitors, failing to exchange with blood progenitors for up to 5 months. Aortic MNPs are different from either typical DCs or macrophages and instead appear to blend distinguishing features of each, more closely resembling macrophages. Using our characterization data, we hope to develop an inducible cell-tracking and a cell-depletion model to differentiate the function of aortic MNPs during the progression of atherosclerosis. Overall, we anticipate these functional analyses will reveal, for the first time, the role of resident aortic MNPs in atherosclerosis.

Abstract 2: Aorta Intima-Resident Macrophages Contribute to Atherosclerotic Lesion Initiation

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Jesse W Williams ◽  
Brian T Saunders ◽  
Ki-Wook Kim ◽  
Slava Epelman ◽  
Kory Lavine ◽  
...  
Keyword(s):  
Atherosclerotic Lesion ◽  
Kinetic Studies ◽  
Endothelial Damage ◽  
Lineage Tracing ◽  
Cellular Mechanisms ◽  
Gm Csf ◽  
Endothelial Surface ◽  
En Face ◽  
Aortic Intima

Atherosclerosis is an underlying cause of cardiovascular disease and a leading cause of mortality worldwide. Macrophage accumulation in atherosclerotic plaque, their uptake of cholesterol, and subsequent local death drive disease progression. Lipid-laden plaque macrophages are thought to be exclusively derived from blood monocyte progenitors that are recruited following endothelial damage induced by cholesterol exposure. In our current study, we focused on characterization of resident vascular macrophages that reside in the aortic intima in plaque-prone areas, previously identified as ‘vascular dendritic cells’. Using en face whole-mount confocal microscopy of aortas, we confirm a uniform resident CD64 + CD11c + CX3CR1 + MHCII + macrophage population, which is present in C57/BL6 mice resistant to atherosclerosis. Importantly, they do not express dendritic cell restricted genes zBTB46 or L-myc. We find aortic macrophages require M-CSF and Flt3 signaling for survival, but are independent of CCR2, CCR7, and GM-CSF receptor signaling, making them a distinct myeloid population. Lineage-tracing and parabiosis approaches suggest these cells derive from definitive hematopoiesis and are then self-maintained independent of blood-progenitors. Using these characterization data, we developed a labeling strategy to identify resident from recruited macrophages during kinetic studies of lesion progression. We find that resident aortic macrophages are the first cells to take up lipid following high fat diet exposure and expand within the arterial wall to form the initial lesion bed. In the absence of resident macrophages early lipid deposition in the aortic arch is ablated. Finally, utilizing an intravital carotid artery imaging approach, we identify resident aortic macrophages to be potential mediators of monocyte recruitment through direct interactions with rolling monocytes on the endothelial surface under diseased and steady-states. Overall, these results shift our understanding of the cellular mechanisms responsible for plaque construction and maintenance.

scholarly journals Selective and inducible targeting of CD11b+mononuclear phagocytes in the murine lung with hCD68-rtTA transgenic systems

2016 ◽  
Vol 311 (1) ◽  
pp. L87-L100 ◽  
Author(s):  
Alexandra L. McCubbrey ◽  
Lea Barthel ◽  
Kara J. Mould ◽  
Michael P. Mohning ◽  
Elizabeth F. Redente ◽  
...  
Keyword(s):  
Lineage Tracing ◽  
Mononuclear Phagocytes ◽  
Acute Lung Inflammation ◽  
Unique Role ◽  
Murine Lung ◽  
Disease States ◽  
Reporter Mice ◽  
Gfp Reporter ◽  
Low Levels

During homeostasis two distinct macrophage (Mø) populations inhabit the lungs: tissue Mø (often called interstitial Mø) and resident alveolar Mø (resAMø). During acute lung inflammation, monocytes from the circulation migrate to areas of injury where they mature into a third Mø population: recruited Mø. Resident AMø uniquely express low levels of CD11b and high levels of CD11c. In comparison, recruited Mø and tissue Mø express high levels of CD11b and low levels of CD11c. It is likely that these three Mø subpopulations play distinct roles in injury and disease states; however, tools with which to individually target or track these populations are lacking. Here we demonstrate the utility of an hCD68-rtTA transgenic system for specific, robust, and inducible targeting of CD11b+recruited Mø and tissue Mø in the murine lung with negligible activation in resAMø. Using hCD68rtTA-GFP reporter mice, we show both during homeostasis and inflammation that administration of doxycycline induces tet-On reporter expression in recruited Mø and tissue Mø but not in resident AMø. We further demonstrate how hCD68-rtTA can be effectively combined with tet-On Cre to target these same recMø and tissue Mø. Accordingly, the hCD68-rtTA system is a powerful new tool that can be used for lineage tracing, fate mapping, and gene deletion in a variety of murine models, thereby enabling sophisticated investigation of the unique role of these CD11b+Mø during lung heath and disease.

scholarly journals MEDALT: single-cell copy number lineage tracing enabling gene discovery

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Fang Wang ◽  
Qihan Wang ◽  
Vakul Mohanty ◽  
Shaoheng Liang ◽  
Jinzhuang Dou ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Single Cell ◽  
Copy Number ◽  
Speciation Analysis ◽  
Population Based ◽  
Lineage Tracing ◽  
Breast Cancer Patients ◽  
Lineage Tree ◽  
History Of ◽  
A Cell

AbstractWe present a Minimal Event Distance Aneuploidy Lineage Tree (MEDALT) algorithm that infers the evolution history of a cell population based on single-cell copy number (SCCN) profiles, and a statistical routine named lineage speciation analysis (LSA), whichty facilitates discovery of fitness-associated alterations and genes from SCCN lineage trees. MEDALT appears more accurate than phylogenetics approaches in reconstructing copy number lineage. From data from 20 triple-negative breast cancer patients, our approaches effectively prioritize genes that are essential for breast cancer cell fitness and predict patient survival, including those implicating convergent evolution.The source code of our study is available at https://github.com/KChen-lab/MEDALT.

scholarly journals Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

2002 ◽  
Vol 68 (9) ◽  
pp. 4390-4398 ◽  
Author(s):  
S. A. F. T. van Hijum ◽  
G. H. van Geel-Schutten ◽  
H. Rahaoui ◽  
M. J. E. C. van der Maarel ◽  
L. Dijkhuizen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Lactobacillus Reuteri ◽  
Bacterial Strains ◽  
Potential Health ◽  
Isolation And Characterization ◽  
A Cell ◽  
Encoding Gene ◽  
First Time

ABSTRACT Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>107) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.

TAMI-36. TAMEP ARE BRAIN TUMOR PARENCHYMAL CELLS CONTROLLING NEOPLASTIC ANGIOGENESIS AND PROGRESSION

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi205-vi206
Author(s):  
Roland Kälin ◽  
Linzhi Cai ◽  
Yuping Li ◽  
Ines Hellmann ◽  
Rainer Glass
Keyword(s):  
Brain Tumor ◽  
Brain Tumors ◽  
Single Cell ◽  
Disease Progression ◽  
Progenitor Cell ◽  
Local Environment ◽  
Lineage Tracing ◽  
Immunofluorescence Analysis ◽  
Progenitor Cell Population ◽  
Parenchymal Cells

Abstract Aggressive brain tumors like glioblastoma depend on support by their local environment and subsets of tumor-parenchymal cells may promote specific phases of disease-progression. We investigated the glioblastoma microenvironment with transgenic lineage-tracing models, intravital imaging, single-cell transcriptomics, immunofluorescence analysis as well as histopathology and characterized a previously unacknowledged population of tumor-associated cells with a myeloid-like expression profile (TAMEP) that transiently appeared during glioblastoma growth. TAMEP of mice and humans were identified with specific markers. Strikingly, TAMEP did not derive from microglia or peripheral monocytes but were generated by a fraction of CNS-resident, SOX2-positive progenitors. Abrogation of this progenitor cell-population, by conditional Sox2-knockout, drastically reduced glioblastoma-vascularization and -size. TAMEP manipulation profoundly altered vessel function and strongly attenuated the blood-tumor barrier. Hence, our data indicate TAMEP and their progenitors as new targets for glioblastoma therapy.

Abstract 120: Pericytes Contribute To Myofibroblast Expansion And Vascular Maturation In The Infarcted Heart

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Linda Alex ◽  
Kai Su ◽  
Izabela Tuleta ◽  
Nikolaos G Frangogiannis
Keyword(s):  
Lineage Tracing ◽  
Cytokine Gene Expression ◽  
Infarct Zone ◽  
Mural Cell ◽  
Infarcted Myocardium ◽  
Reporter Mice ◽  
Infarct Healing ◽  
Subsequent Activation ◽  
Vascular Maturation

Infarct healing is dependent on recruitment of inflammatory leukocytes and subsequent activation of myofibroblasts (MF) and neovessel formation, ultimately resulting in formation of a highly vascularized collagen-enriched scar. Though the heart has an abundant population of periendothelial pericytes, its role in wound healing upon myocardial infarction (MI) has not been studied. We hypothesized that in the infarcted myocardium, pericytes may become activated, contributing to inflammatory, fibrotic and angiogenic responses. We used pericyte/fibroblast reporter mice (NG2 DsRed ;PDGFRα GFP ), lineage tracing studies and in vitro approaches to study the fate and role of pericytes in the infarcted myocardium. In normal hearts, NG2+/PDGFRα- pericytes and PDGFRα+/NG2- fibroblasts had distinct transcriptomic profiles. Pericytes expressed mural genes like Acta2 , Pdgfrb and low amounts of extracellular matrix (ECM) genes, whereas fibroblasts synthesized collagens, Timp2/3 and matricellular genes. 7 days post-MI, expansion of the NG2+ population in the infarct zone was associated with emergence of non-mural NG2+/αSMA+ cells with MF characteristics. FACS-sorted NG2+/PDGFRα- cells from 7-day infarcts expressed higher levels of collagens when compared to NG2+/PDGFRα- cells from normal hearts. Infarct pericytes had high integrin and MMP14 expression, reflecting an activated migratory phenotype. Lineage tracing using NG2CreER TM ;Rosa tdTomato ;PDGFRα GFP mice showed that 5.7%±1.04 of PDGFRα+ fibroblasts and 10.49%±2.73 of infarct MFs were derived from NG2+ lineage. Pericyte-derived fibroblasts exhibited higher ECM gene synthesis, in comparison to fibroblasts from non-pericyte origin, while pericyte-derived mural cells showed accentuated inflammatory cytokine gene expression. Immunostaining showed pericytes actively contribute to vascular maturation, forming a mural cell coat enwrapping infarct neovessels. In vitro, TGFβ induced integrins, collagens and MMPs in human pericytes, similar to the changes observed in infarct pericytes. Taken together, our evidences show that after MI, pericytes become activated and contribute to repair by undergoing conversion to a subset of myofibroblasts and by coating infarct neovessels.

scholarly journals Macrophage Autophagy and Oxidative Stress: An Ultrastructural and Immunoelectron Microscopical Study

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Ida Perrotta ◽  
Valentina Carito ◽  
Emilio Russo ◽  
Sandro Tripepi ◽  
Saveria Aquila ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Defense Mechanisms ◽  
Ultrastructural Localization ◽  
Microscopical Study ◽  
Beclin 1 ◽  
Cell Organelles ◽  
A Cell ◽  
First Time

The word autophagy broadly refers to the cellular catabolic processes that lead to the removal of damaged cytosolic proteins or cell organelles through lysosomes. Although autophagy is often observed during programmed cell death, it may also serve as a cell survival mechanism. Accumulation of reactive oxygen species within tissues and cells induces various defense mechanisms or programmed cell death. It has been shown that, besides inducing apoptosis, oxidative stress can also induce autophagy. To date, however, the regulation of autophagy in response to oxidative stress remains largely elusive and poorly understood. Therefore, the present study was designed to examine the ratio between oxidative stress and autophagy in macrophages after oxidant exposure (AAPH) and to investigate the ultrastructural localization of beclin-1, a protein essential for autophagy, under basal and stressful conditions. Our data provide evidence that oxidative stress induces autophagy in macrophages. We demonstrate, for the first time by immunoelectron microscopy, the subcellular localization of beclin-1 in autophagic cells.

scholarly journals Detection of Amyotrophic Lateral Sclerosis (ALS) via Acoustic Analysis

2018 ◽  
Author(s):  
Raquel Norel ◽  
Mary Pietrowicz ◽  
Carla Agurto ◽  
Shay Rishoni ◽  
Guillermo Cecchi
Keyword(s):  
Disease Progression ◽  
Acoustic Analysis ◽  
Voice Quality ◽  
Non Invasive ◽  
Frequent Monitoring ◽  
In The Wild ◽  
A Cell ◽  
Speech Features ◽  
Severity Of The Disease ◽  
Lateral Sclerosis

ABSTRACTALS is a fatal neurodegenerative disease with no cure. Experts typically measure disease progression via the ALSFRS-R score, which includes measurements of various abilities known to decline. We propose instead the use of speech analysis as a proxy for ALS progression. This technique enables 1) frequent non-invasive, inexpensive, longitudinal analysis, 2) analysis of data recorded in the wild, and 3) creation of an extensive ALS databank for future analysis. Patients and trained medical professionals need not be co-located, enabling more frequent monitoring of more patients from the convenience of their own homes. The goals of this study are the identification of acoustic speech features in naturalistic contexts which characterize disease progression and development of machine models which can recognize the presence and severity of the disease. We evaluated subjects from the Prize4Life Israel dataset, using a variety of frequency, spectral, and voice quality features. The dataset was generated using the ALS Mobile Analyzer, a cell-phone app that collects data regarding disease progress using a self-reported ALSFRS-R questionnaire and several active tasks that measure speech and motor skills. Classification via leave-five-subjects-out cross-validation resulted in an accuracy rate of 79% (61% chance) for males and 83% (52% chance) for females.

scholarly journals Selection and Characterization of Rupintrivir-Resistant Norwalk Virus Replicon Cells In Vitro

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Mitsutaka Kitano ◽  
Myra Hosmillo ◽  
Edward Emmott ◽  
Jia Lu ◽  
Ian Goodfellow
Keyword(s):  
Reverse Genetics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Murine Norovirus ◽  
Norwalk Virus ◽  
Irreversible Inhibitor ◽  
Antiviral Compounds ◽  
A Cell ◽  
First Time

ABSTRACT Human norovirus (HuNoV) is a major cause of nonbacterial gastroenteritis worldwide, yet despite its impact on society, vaccines and antivirals are currently lacking. A HuNoV replicon system has been widely applied to the evaluation of antiviral compounds and has thus accelerated the process of drug discovery against HuNoV infection. Rupintrivir, an irreversible inhibitor of the human rhinovirus 3C protease, has been reported to inhibit the replication of the Norwalk virus replicon via the inhibition of the norovirus protease. Here we report, for the first time, the generation of rupintrivir-resistant human Norwalk virus replicon cells in vitro . Sequence analysis revealed that these replicon cells contained amino acid substitutions of alanine 105 to valine (A105V) and isoleucine 109 to valine (I109V) in the viral protease NS6. The application of a cell-based fluorescence resonance energy transfer (FRET) assay for protease activity demonstrated that these substitutions were involved in the enhanced resistance to rupintrivir. Furthermore, we validated the effect of these mutations using reverse genetics in murine norovirus (MNV), demonstrating that a recombinant MNV strain with a single I109V substitution in the protease also showed reduced susceptibility to rupintrivir. In summary, using a combination of different approaches, we have demonstrated that, under the correct conditions, mutations in the norovirus protease that lead to the generation of resistant mutants can rapidly occur.

scholarly journals B Cell Specific Knockdown of the Erythropoietin (EPO) Receptor Attenuates EPO-Induced Bone Loss in Mice

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 939-939
Author(s):  
Albert Kolomansky ◽  
Naamit Deshet-Unger ◽  
Nathalie Ben-Califa ◽  
Zamzam Awida ◽  
Maria Ibrahim ◽  
...  
Keyword(s):  
B Cells ◽  
Bone Loss ◽  
B Cell ◽  
Trabecular Bone ◽  
Cell Lineage ◽  
Lineage Tracing ◽  
Trabecular Bone Loss ◽  
Β3 Integrin ◽  
First Time

Background and aims: Erythropoietin (EPO) is the key regulator of red blood cell production, commonly used in clinical practice to treat certain forms of anemia. Our studies and those of others have demonstrated that EPO administration induces substantial trabecular bone loss. We proposed that EPO-induced bone loss is partially mediated by subsets of bone marrow (BM) B cells that express EPO-R. Mechanistically, EPO upregulates the surface expression of RANKL by BM B cells and augments B cell-derived osteoclastogenesis in vitro. We showed that the latter is likely mediated by pro-B cells expressing the MCS-F receptor (CD115) and capable of transdifferentiation to osteoclasts (Abstract # 1007, EHA 2017). Here we address the role of B cell-specific EPO-R in EPO-induced bone loss (i.e. at supra-physiological EPO levels). Moreover, we demonstrate, for the first time, the occurrence of B cell-derived osteoclastogenesis in vivo, a finding of critical importance in the field of osteohematology. Methods: In order to trace the B cell lineage from its earliest precursors, we used the MB1-Cre mouse line combined with either the R26R-EYFP or the EPO-Rfl/fl mice for lineage tracing and B cell-specific EPO-R knockdown, respectively. Sequential fluorescence and light microscopy were used for the demonstration of B cell-derived osteoclastogenesis in vivo. Human recombinant EPO was administered in vivo at a dose of 180IU thrice weekly for two weeks. Immunophenotyping of BM B cell populations was assessed by multi-color flow cytometry. Results: Using female MB1-Cre; EPO-Rfl/fl (cKD) mice, we found that B cell-specific EPO-R knockdown attenuated the profound EPO-induced trabecular bone loss in the proximal part of the femoral distal metaphysis (proximal BV/TV 0.034±0.012% vs 0.007±0.003% in the cKD vs control mice, p<0.05, Figure 1). Remarkably, this effect was observed despite the fact that cKD mice attained higher hemoglobin levels following EPO treatment (21.1±0.1 mg/dL vs 20.4±0.2 mg/dL in the cKD vs control mice, p<0.05). An EPO-induced increase in CD115+ Pro-B cells was observed in EPO-treated control mice but was absent in the cKD mice. The latter finding correlates with the observed bone loss and indicates that the increased number of MCSF-R-expressing pro-B cells is dependent on B cell EPO-R. Supporting the osteoclastic potential of this specific B cell subpopulation is the fact that most of the CD115+ Pro-B cells also express β3 integrin (CD61) which is essential for osteoclast differentiation and function. Using the MB1-Cre;R26R-EYFP murine model for B cell lineage tracing, we could demonstrate that some of the TRAP+/ β3 integrin+ bone lining cells were also positive for EYFP (Figure 2). This demonstrates the B cell origin of some of the osteoclasts in vivo. Conclusions: Our work highlights B cells as an important extra-erythropoietic target of EPO-EPO-R signaling that regulates bone homeostasis and might also indirectly affect EPO-stimulated erythropoietic response. The relevance and the mechanisms of the latter phenomenon merits further investigation. Importantly, we present here, for the first time, histological evidence for B cell-derived osteoclastogenesis in vivo, thus opening novel research avenues. DN and YG Equal contribution Funded by the German Israel Foundation, Grant # 01021017 to YG, DN, MR and BW and by the Israel Science Foundation (ISF) Grant No. 343/17 to DN. Disclosures Mittelman: Novartis: Honoraria, Research Funding, Speakers Bureau.

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