ABSTRACT
The developing mouse pancreas is surrounded by mesoderm compartments providing signals that induce pancreas formation. Most pancreatic organoid protocols lack this mesoderm niche and only partially capture the pancreatic cell repertoire. This work aims to generate pancreatic aggregates by differentiating mouse embryonic stem cells (mESCs) into mesoderm progenitors (MPs) and pancreas progenitors (PPs), without using Matrigel. First, mESCs were differentiated into epiblast stem cells (EpiSCs) to enhance the PP differentiation rate. Next, PPs and MPs aggregated together giving rise to various pancreatic cell types, including endocrine, acinar, and ductal cells, and to endothelial cells. Single-cell RNA sequencing analysis revealed a larger endocrine population within the PP + MP aggregates, as compared to PPs alone or PPs in Matrigel aggregates. The PP + MP aggregate gene expression signatures and its endocrine population percentage closely resembled those of the endocrine population found in the mouse embryonic pancreas, which holds promise for studying pancreas development.
ABSTRACT
BACKGROUND: Breast cancer clinical outcome relies on its intrinsic molecular subtype and mortality is almost exclusively due to metastasis, whose mechanism remains unclear. We recently revealed the specific contribution of plasma membrane cholesterol to the invasion of malignant MCF10CAIa but not premalignant MCF10AT and normal MCF10A cell lines in 2D, through invadopodia formation and extracellular matrix (ECM) degradation. In the present study, we address the impact of breast cancer subtypes, mutations and aggressiveness on cholesterol implication in breast cancer cell invasion and 3D spheroid invasion and growth. METHODS: We used nine breast cancer cell lines grouped in four subtypes matching breast tumor classification. Four of these cell lines were also used to generate 3D spheroids. These cell lines were compared for cell invasion in 2D and 3D, spheroid growth in 3D, gelatin degradation, cortactin expression, activation and subcellular distribution as well as cell surface cholesterol distribution and lipid droplets. The effect of plasma membrane cholesterol depletion on all these parameters was determined in parallel and systematically compared with the impact of global matrix metalloproteinase (MMP) inhibition. RESULTS: The six invasive cell lines in 2D were sensitive to partial cholesterol depletion, independently of their subtype, aggressiveness or mutation. Nevertheless, the effect was stronger in the three cell lines able to degrade gelatin. 3D spheroid invasion was also reduced after cholesterol depletion in all breast cancer subtypes tested. Notably, targeting cholesterol was more powerful than MMP inhibition in reducing invasion in both 2D and 3D culture models. Moreover, cholesterol depletion in the six invasive cell lines impaired cortactin distribution in the perinuclear region where invadopodia localized. Breast cancer cell line aggressiveness relied on cholesterol-enriched domains at the ECM-free side and intracellular lipid droplets. Furthermore, the three gelatin-degrading cell lines were characterized by increased cholesterol-enriched submicrometric domains at their ECM-contact side. CONCLUSION: Together, our data suggest cell surface cholesterol combined with lipid droplet labeling as a breast cancer cell aggressiveness marker. They also open the way to test other cholesterol-targeting drugs in more complex models to further evaluate whether cholesterol could represent a strategy in breast cancer therapy.
ABSTRACT
A specific plasma membrane distribution of the mechanosensitive ion channel Piezo1 is required for cell migration, but the mechanism remains elusive. Here, we addressed this question using WT and Piezo1-silenced C2C12 mouse myoblasts and WT and Piezo1-KO human kidney HEK293T cells. We showed that cell migration in a cell-free area and through a porous membrane decreased upon Piezo1 silencing or deletion, but increased upon Piezo1 activation by Yoda1, whereas migration towards a chemoattractant gradient was reduced by Yoda1. Piezo1 organized into clusters, which were preferentially enriched at the front. This polarization was stimulated by Yoda1, accompanied by Ca2+ polarization, and abrogated by partial cholesterol depletion. Piezo1 clusters partially colocalized with cholesterol- and GM1 ganglioside-enriched domains, the proportion of which was increased by Yoda1. Mechanistically, Piezo1 activation induced a differential mobile fraction of GM1 associated with domains and the bulk membrane. Conversely, cholesterol depletion abrogated the differential mobile fraction of Piezo1 associated with clusters and the bulk membrane. In conclusion, we revealed, for the first time, the differential implication of Piezo1 depending on the migration mode and the interplay between GM1/cholesterol-enriched domains at the front during migration in a cell-free area. These domains could provide the optimal biophysical properties for Piezo1 activity and/or spatial dissociation from the PMCA calcium efflux pump.
Subject(s)
G(M1) Ganglioside , Ion Channels , Animals , Humans , Mice , Cell Movement , Cholesterol , HEK293 Cells , Ion Channels/metabolismABSTRACT
[This corrects the article DOI: 10.3389/fphys.2023.1205493.].
ABSTRACT
Estradiol and progesterone are key regulators of the menstrual cycle. In the human endometrium, progesterone induces morphological changes required for blastocyst implantation. Dysregulated response to progesterone can lead to endometrial pathologies including uterine bleeding and endometriosis. Besides the canonical nuclear progesterone receptor (encoded by the PGR gene), alternative response pathways include Progesterone Receptor Membrane Component 1 (PGRMC1), suspected to be involved in pathogenesis of endometrial diseases. We previously reported the spatiotemporal profile of PGRMC1 expression in the human endometrium along the menstrual cycle, highlighting progressive increase and decrease during the proliferative and secretory phases, respectively. Here we directly addressed its regulation by estradiol and progesterone, with systematic comparison with regulation of PGR expression. We found a direct correlation between expression of both genes during the proliferative and secretory phases in the cycling endometrium, but not during the menstrual phase. In a xenograft model mimicking the cycle phases, estradiol significantly increased and progesterone significantly decreased PGR expression but changes were not significant for PGRMC1. Finally, we did not find any significant effect of the ovarian steroids on expression of PGR or PGRMC1 in primary culture of endometrial stromal cells, except for a small increase in PGR expression by estradiol. Altogether, our experiments do not allow a major advance in our understanding of the mechanisms of cyclic variation of PGRMC1 expression, in particular regarding potential regulation by the ovarian steroids.
Subject(s)
Progesterone , Receptors, Progesterone , Female , Humans , Progesterone/pharmacology , Progesterone/metabolism , Receptors, Progesterone/genetics , Receptors, Progesterone/metabolism , Estradiol/pharmacology , Estradiol/metabolism , Heterografts , Endometrium/pathology , Steroids/metabolism , Cell Culture Techniques , Membrane Proteins/genetics , Membrane Proteins/metabolismABSTRACT
Transfusion of red blood cell concentrates is the most common medical procedure to treat anaemia. However, their storage is associated with development of storage lesions, including the release of extracellular vesicles. These vesicles affect in vivo viability and functionality of transfused red blood cells and appear responsible for adverse post-transfusional complications. However, the biogenesis and release mechanisms are not fully understood. We here addressed this issue by comparing the kinetics and extents of extracellular vesicle release as well as red blood cell metabolic, oxidative and membrane alterations upon storage in 38 concentrates. We showed that extracellular vesicle abundance increased exponentially during storage. The 38 concentrates contained on average 7 × 1012 extracellular vesicles at 6 weeks (w) but displayed a â¼40-fold variability. These concentrates were subsequently classified into 3 cohorts based on their vesiculation rate. The variability in extracellular vesicle release was not associated with a differential red blood cell ATP content or with increased oxidative stress (in the form of reactive oxygen species, methaemoglobin and band3 integrity) but rather with red blood cell membrane modifications, i.e., cytoskeleton membrane occupancy, lateral heterogeneity in lipid domains and transversal asymmetry. Indeed, no changes were noticed in the low vesiculation group until 6w while the medium and the high vesiculation groups exhibited a decrease in spectrin membrane occupancy between 3 and 6w and an increase of sphingomyelin-enriched domain abundance from 5w and of phosphatidylserine surface exposure from 8w. Moreover, each vesiculation group showed a decrease of cholesterol-enriched domains associated with a cholesterol content increase in extracellular vesicles but at different storage time points. This observation suggested that cholesterol-enriched domains could represent a starting point for vesiculation. Altogether, our data reveal for the first time that the differential extent of extracellular vesicle release in red blood cell concentrates did not simply result from preparation method, storage conditions or technical issues but was linked to membrane alterations.
ABSTRACT
The pancreas is a complex organ consisting of differentiated cells and extracellular matrix (ECM) organized adequately to enable its endocrine and exocrine functions. Although much is known about the intrinsic factors that control pancreas development, very few studies have focused on the microenvironment surrounding pancreatic cells. This environment is composed of various cells and ECM components, which play a critical role in maintaining tissue organization and homeostasis. In this study, we applied mass spectrometry to identify and quantify the ECM composition of the developing pancreas at the embryonic (E) day 14.5 and postnatal (P) day 1 stages. Our proteomic analysis identified 160 ECM proteins that displayed a dynamic expression profile with a shift in collagens and proteoglycans. Furthermore, we used atomic force microscopy to measure the biomechanical properties and found that the pancreatic ECM was soft (≤400 Pa) with no significant change during pancreas maturation. Lastly, we optimized a decellularization protocol for P1 pancreatic tissues, incorporating a preliminary crosslinking step, which effectively preserved the 3D organization of the ECM. The resulting ECM scaffold proved suitable for recellularization studies. Our findings provide insights into the composition and biomechanics of the pancreatic embryonic and perinatal ECM, offering a foundation for future studies investigating the dynamic interactions between the ECM and pancreatic cells.
Subject(s)
Proteomics , Tissue Engineering , Tissue Engineering/methods , Proteomics/methods , Extracellular Matrix/metabolism , Pancreas/metabolism , Extracellular Matrix Proteins/metabolism , Pancreatic Hormones/metabolism , Tissue Scaffolds/chemistryABSTRACT
Myoblast migration is crucial for myogenesis and muscular tissue homeostasis. However, its spatiotemporal control remains elusive. Here, we explored the involvement of plasma membrane cholesterol and sphingolipids in this process. In resting C2C12 mouse myoblasts, those lipids clustered in sphingomyelin/cholesterol/GM1 ganglioside (SM/chol/GM1)- and cholesterol (chol)-enriched domains, which presented a lower stiffness than the bulk membrane. Upon migration, cholesterol and sphingomyelin polarized at the front, forming cholesterol (chol)- and sphingomyelin/cholesterol (SM/chol)-enriched domains, while GM1-enriched domains polarized at the rear. A comparison of domain proportion suggested that SM/chol- and GM1-enriched domains originated from the SM/chol/GM1-coenriched domains found at resting state. Modulation of domain proportion (through cholesterol depletion, combined or not with actin polymerization inhibition, or sphingolipid synthesis inhibition) revealed that the higher the chol- and SM/chol-enriched domains, the higher the myoblast migration. At the front, chol- and SM/chol-enriched domains were found in proximity with F-actin fibers and the lateral mobility of sphingomyelin in domains was specifically restricted in a cholesterol- and cytoskeleton-dependent manner while domain abrogation impaired F-actin and focal adhesion polarization. Altogether, we showed the polarization of cholesterol and sphingomyelin and their clustering in chol- and SM/chol-enriched domains with differential properties and roles, providing a mechanism for the spatial and functional control of myoblast migration.
Subject(s)
G(M1) Ganglioside , Sphingomyelins , Animals , Mice , Sphingomyelins/metabolism , Actins , Cholesterol/metabolism , Cluster AnalysisABSTRACT
PURPOSE: This study aimed to investigate the modulation of circulating exosome-like extracellular vesicles (ELVs) after 6 wk of sprint interval training (SIT) at sea level and at 2000, 3000, and 4000 m. METHODS: Thirty trained endurance male athletes (18-35 yr) participated in a 6-wk SIT program (30-s all-out sprint, 4-min 30-s recovery; 4-9 repetitions, 2 sessions per week) at sea level ( n = 8), 2000 m (fraction of inspired oxygen (F io2 ) 0.167, n = 8), 3000 m (F io2 0.145, n = 7), or 4000 m (F io2 0.13, n = 7). Venous blood samples were taken before and after the training period. Plasma ELVs were isolated by size exclusion chromatography, counted by nanoparticle tracking analysis, and characterized according to international standards. Candidate ELV microRNAs (miRNAs) were quantified by real-time polymerase chain reaction. RESULTS: When the three hypoxic groups were analyzed separately, only very minor differences could be detected in the levels of circulating particles, ELV markers, or miRNA. However, the levels of circulating particles increased (+262%) after training when the three hypoxic groups were pooled, and tended to increase at sea level (+65%), with no difference between these two groups. A trend to an increase was observed for the two ELV markers, TSG101 (+65%) and HSP60 (+441%), at sea level, but not in hypoxia. Training also seemed to decrease the abundance of miR-23a-3p and to increase the abundance of miR-21-5p in hypoxia but not at sea level. CONCLUSIONS: A 6-wk SIT program tended to increase the basal levels of circulating ELVs when performed at sea level but not in hypoxia. In contrast, ELV miRNA cargo seemed to be modulated in hypoxic conditions only. Further research should explore the potential differences in the origin of ELVs between normoxic and local and systemic hypoxic conditions.
Subject(s)
Extracellular Vesicles , High-Intensity Interval Training , MicroRNAs , Humans , Male , Altitude , Exosomes , Hypoxia , Adolescent , Young Adult , AdultABSTRACT
Piezo1 is a mechanosensitive ion channel required for various biological processes, but its regulation remains poorly understood. Here, we used erythrocytes to address this question since they display Piezo1 clusters, a strong and dynamic cytoskeleton and three types of submicrometric lipid domains, respectively enriched in cholesterol, GM1 ganglioside/cholesterol and sphingomyelin/cholesterol. We revealed that Piezo1 clusters were present in both the rim and the dimple erythrocyte regions. Upon Piezo1 chemical activation by Yoda1, the Piezo1 cluster proportion mainly increased in the dimple area. This increase was accompanied by Ca2+ influx and a rise in echinocytes, in GM1/cholesterol-enriched domains in the dimple and in cholesterol-enriched domains in the rim. Conversely, the effects of Piezo1 activation were abrogated upon membrane cholesterol depletion. Furthermore, upon Piezo1-independent Ca2+ influx, the above changes were not observed. In healthy donors with a high echinocyte proportion, Ca2+ influx, lipid domains and Piezo1 fluorescence were high even at resting state, whereas the cytoskeleton membrane occupancy was lower. Accordingly, upon decreases in cytoskeleton membrane occupancy and stiffness in erythrocytes from patients with hereditary spherocytosis, Piezo1 fluorescence was increased. Altogether, we showed that Piezo1 was differentially controlled by lipid domains and the cytoskeleton and was favored by the stomatocyte-discocyte-echinocyte transformation.
Subject(s)
Cytoskeleton , Ion Channels , Microtubules , Humans , Cholesterol , Erythrocytes , G(M1) Ganglioside , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Ion Channels/metabolismABSTRACT
Extracellular vesicles are spherical subcellular structures delimited by a lipid bilayer and released by most cells in the human body. They are loaded with a myriad of molecules (i.e., nucleic acids and proteins) depending on their cell of origin and provide the ability to transmit a message to surrounding or distant target cells. In several organs, including the thyroid, abundant recent literature reports that extracellular vesicles are responsible for intercellular communication in physiological and pathological processes, and that their utilization as a potential biomarker of pathological states (i.e., cancer, autoimmune diseases) or as therapeutic delivery vehicles promise clinical options. In this review, we present the current knowledge and understanding regarding the role of extracellular vesicles in developing thyroid diseases and diagnosis.
ABSTRACT
Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAFV600E genetic alteration is found in 60% of this endocrine cancer. This oncogene is associated with poor prognosis, resistance to radioiodine therapy, and tumor progression. Histological follow-up by anatomo-pathologists revealed that two-thirds of surgically-removed thyroids do not present malignant lesions. Thus, continued fundamental research into the molecular mechanisms of TC downstream of BRAFV600E remains central to better understanding the clinical behavior of these tumors. To study PTC, we used a mouse model in which expression of BRAFV600E was specifically switched on in thyrocytes by doxycycline administration. Upon daily intraperitoneal doxycycline injection, thyroid tissue rapidly acquired histological features mimicking human PTC. Transcriptomic analysis revealed major changes in immune signaling pathways upon BRAFV600E induction. Multiplex immunofluorescence confirmed the abundant recruitment of macrophages, among which a population of LYVE-1+/CD206+/STABILIN-1+ was dramatically increased. By genetically inactivating the gene coding for the scavenger receptor STABILIN-1, we showed an increase of CD8+ T cells in this in situ BRAFV600E-dependent TC. Lastly, we demonstrated the presence of CD206+/STABILIN-1+ macrophages in human thyroid pathologies. Altogether, we revealed the recruitment of immunosuppressive STABILIN-1 macrophages in a PTC mouse model and the interest to further study this macrophage subpopulation in human thyroid tissues.
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Development of the pancreas is driven by an intrinsic program coordinated with signals from other cell types in the epithelial environment. These intercellular communications have been so far challenging to study because of the low concentration, localized production and diversity of the signals released. Here, we combined scRNAseq data with a computational interactomic approach to identify signals involved in the reciprocal interactions between the various cell types of the developing pancreas. This in silico approach yielded 40,607 potential ligand-target interactions between the different main pancreatic cell types. Among this vast network of interactions, we focused on three ligands potentially involved in communications between epithelial and endothelial cells. BMP7 and WNT7B, expressed by pancreatic epithelial cells and predicted to target endothelial cells, and SEMA6D, involved in the reverse interaction. In situ hybridization confirmed the localized expression of Bmp7 in the pancreatic epithelial tip cells and of Wnt7b in the trunk cells. On the contrary, Sema6d was enriched in endothelial cells. Functional experiments on ex vivo cultured pancreatic explants indicated that tip cell-produced BMP7 limited development of endothelial cells. This work identified ligands with a restricted tissular and cellular distribution and highlighted the role of BMP7 in the intercellular communications contributing to vessel development and organization during pancreas organogenesis.
Subject(s)
Endothelial Cells , Organogenesis , Cell Differentiation/physiology , Endothelial Cells/metabolism , Ligands , Organogenesis/physiology , Pancreas/metabolismABSTRACT
Differential diagnosis of thyroid cancer and benign nodules is still one of the most challenging issues in the field of endocrinology. To overcome overdiagnosis of papillary thyroid carcinomas (PTC) and the consecutive overtreatment of multinodular diseases, the search for easily accessible, sensitive and accurate biomarkers is critical. Several micro-RNAs (miRNAs) freely circulating in peripheral blood or enclosed in extracellular vesicles (EVs) have been proposed as potential biomarkers from non-invasive liquid biopsies. However, protocols are rarely comparable and conflicting data exist in the literature. In this work, we aimed to assess the diagnostic value of six micro-RNAs by comparing their expression in thyroid tissue to their abundance in bulk plasma and in plasma-EVs, before and after thyroid surgery. Plasma-EVs were isolated using a sequential density- and size-based fractionation, followed by in-depth characterization, confirming EV purity. Micro-RNA levels were measured by RT-qPCR in thyroid tissue, plasma and plasma-EVs. Among the six candidates, only miR-146b-5p and miR-21a-5p displayed a significant differential abundance in purified plasma-derived EVs from patients with PTC and benign disease. However, no difference could be demonstrated in bulk plasma through our cohort of patients. Overall, our work supports the use of a well-defined protocol of plasma-EV miRNAs purification for biomarker discovery, rather than the use of freely circulating miRNAs in bulk plasma. Our work also demonstrates that standardized pre-analytical and analytical procedures as well as optimized EV-miRNAs detection methods are essential.
Subject(s)
Extracellular Vesicles , MicroRNAs , Thyroid Neoplasms , Biomarkers , Biomarkers, Tumor/metabolism , Extracellular Vesicles/metabolism , Humans , MicroRNAs/genetics , Thyroid Cancer, Papillary/metabolism , Thyroid Neoplasms/pathologyABSTRACT
Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment.
ABSTRACT
While the signaling pathways and transcription factors involved in the differentiation of thyroid follicular cells, both in embryonic and adult life, are increasingly well understood, the underlying mechanisms and potential crosstalk between the thyroid transcription factors Nkx2.1, Foxe1 and Pax8 and inductive signals remain unclear. Here, we focused on the transcription factor Sox9, which is expressed in Nkx2.1-positive embryonic thyroid precursor cells and is maintained from embryonic development to adulthood, but its function and control are unknown. We show that two of the main signals regulating thyroid differentiation, TSH and TGFß, modulate Sox9 expression. Specifically, TSH stimulates the cAMP/PKA pathway to transcriptionally upregulate Sox9 mRNA and protein expression, a mechanism that is mediated by the binding of CREB to a CRE site within the Sox9 promoter. Contrastingly, TGFß signals through Smad proteins to inhibit TSH-induced Sox9 transcription. Our data also reveal that Sox9 transcription is regulated by the thyroid transcription factors, particularly Pax8. Interestingly, Sox9 significantly increased the transcriptional activation of Pax8 and Foxe1 promoters and, consequently, their expression, but had no effect on Nkx2.1. Our study establishes the involvement of Sox9 in thyroid follicular cell differentiation and broadens our understanding of transcription factor regulation of thyroid function.
Subject(s)
SOX9 Transcription Factor/metabolism , Thyroid Epithelial Cells/cytology , Thyroid Epithelial Cells/metabolism , Thyroid Gland/metabolism , Thyrotropin/metabolism , Transcription Factors/metabolism , Transforming Growth Factor beta/metabolism , Animals , Cell Differentiation , Cell Line , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation , Mice , PAX8 Transcription Factor/genetics , PAX8 Transcription Factor/metabolism , Promoter Regions, Genetic , SOX9 Transcription Factor/genetics , Signal Transduction , Thyroid Gland/cytology , Thyroid Gland/embryology , Thyroid Nuclear Factor 1/genetics , Thyroid Nuclear Factor 1/metabolism , Thyrotropin/pharmacology , Transcription, Genetic , Transforming Growth Factor beta/pharmacologyABSTRACT
Generating comprehensive image maps, while preserving spatial three-dimensional (3D) context, is essential in order to locate and assess quantitatively specific cellular features and cell-cell interactions during organ development. Despite recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on two-dimensional histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in three dimensions and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal and endothelial cells) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, whereas the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirements for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as the Pancreas Embryonic Cell Atlas).
Subject(s)
Imaging, Three-Dimensional/methods , Pancreas/anatomy & histology , Animals , Embryo, Mammalian/anatomy & histology , Embryonic Development , Endothelial Cells/cytology , Endothelial Cells/metabolism , Epithelium/anatomy & histology , Homeobox Protein Nkx-2.5/deficiency , Homeobox Protein Nkx-2.5/genetics , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, FluorescenceABSTRACT
The purpose of this study is to investigate exosome-like vesicle (ELV) plasma concentrations and markers of multivesicular body (MVB) biogenesis in skeletal muscle in response to acute exercise. Seventeen healthy [body mass index (BMI): 23.5 ± 0.5 kg·m-2] and 15 prediabetic (BMI: 27.3 ± 1.2 kg·m-2) men were randomly assigned to two groups performing an acute cycling bout in normoxia or hypoxia ([Formula: see text] 14.0%). Venous blood samples were taken before (T0), during (T30), and after (T60) exercise, and biopsies from m. vastus lateralis were collected before and after exercise. Plasma ELVs were isolated by size exclusion chromatography, counted by nanoparticle tracking analysis (NTA), and characterized according to international standards, followed by expression analyses of canonical ELV markers in skeletal muscle. In the healthy normoxic group, the total number of particles in the plasma increased during exercise from T0 to T30 (+313%) followed by a decrease from T30 to T60 (-53%). In the same group, an increase in TSG101, CD81, and HSP60 protein expression was measured after exercise in plasma ELVs; however, in the prediabetic group, the total number of particles in the plasma was not affected by exercise. The mRNA content of TSG101, ALIX, and CD9 was upregulated in skeletal muscle after exercise in normoxia, whereas CD9 and CD81 were downregulated in hypoxia. ELV plasma abundance increased in response to acute aerobic exercise in healthy subjects in normoxia, but not in prediabetic subjects, nor in hypoxia. Skeletal muscle analyses suggested that this tissue did not likely play a major role of the exercise-induced increase in circulating ELVs.
Subject(s)
Exercise , Extracellular Vesicles/metabolism , Hypoxia/blood , Multivesicular Bodies/metabolism , Muscle Contraction , Prediabetic State/blood , Quadriceps Muscle/metabolism , Adult , Bicycling , Calcium-Binding Proteins/blood , Case-Control Studies , Cell Cycle Proteins/blood , DNA-Binding Proteins/blood , Endosomal Sorting Complexes Required for Transport/blood , Humans , Hypoxia/diagnosis , Hypoxia/physiopathology , Male , Middle Aged , Organelle Biogenesis , Prediabetic State/diagnosis , Prediabetic State/physiopathology , Quadriceps Muscle/physiopathology , Random Allocation , Tetraspanin 29/blood , Time Factors , Transcription Factors/bloodABSTRACT
Tight junction complexes are involved in the establishment and maintenance of cell polarity and the regulation of signalling pathways, controlling biological processes such as cell differentiation and cell proliferation. MarvelD3 is a tight junction protein expressed in adult epithelial and endothelial cells. In Xenopus laevis, MarvelD3 morphants present differentiation defects of several ectodermal derivatives. In vitro experiments further revealed that MarvelD3 couples tight junctions to the MEKK1-JNK pathway to regulate cell behaviour and survival. In this work, we found that MarvelD3 is expressed from early developmental stages in the exocrine and endocrine compartments of the pancreas, as well as in endothelial cells of this organ. We thoroughly characterized MarvelD3 expression pattern in developing pancreas and evaluated its function by genetic ablation. Surprisingly, inactivation of MarvelD3 in mice did not alter development and differentiation of the pancreatic tissue. Moreover, tight junction formation and organization, cell polarization, and activity of the JNK-pathway were not impacted by the deletion of MarvelD3.
Subject(s)
MARVEL Domain-Containing Proteins/genetics , Pancreas/embryology , Pancreas/physiology , Tight Junction Proteins/genetics , Animals , CRISPR-Cas Systems , Cell Differentiation/genetics , Female , Gene Expression Regulation, Developmental , MAP Kinase Signaling System/genetics , MARVEL Domain-Containing Proteins/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Pancreas/cytology , Salivary Glands/physiology , Spatio-Temporal Analysis , Tight Junction Proteins/metabolismABSTRACT
Organogenesis is the phase of embryonic development leading to the formation of fully functional organs. In the case of the thyroid, organogenesis starts from the endoderm and generates a multitude of closely packed independent spherical follicular units surrounded by a dense network of capillaries. Follicular organisation is unique and essential for thyroid function, i.e. thyroid hormone production. Previous in vivo studies showed that, besides their nutritive function, endothelial cells play a central role during thyroid gland morphogenesis. However, the precise mechanisms and biological parameters controlling the transformation of the multi-layered thyroid epithelial primordium into a multitude of single-layered follicles are mostly unknown. Animal studies used to improve understanding of organogenesis are costly and time-consuming, with recognised limitations. Here, we developed and used a 2-D vertex model of thyroid growth, angiogenesis and folliculogenesis, within the open-source Chaste framework. Our in silico model, based on in vivo images, correctly simulates the differential growth and proliferation of central and peripheral epithelial cells, as well as the morphogen-driven migration of endothelial cells, consistently with our experimental data. Our simulations further showed that reduced epithelial cell adhesion was critical to allow endothelial invasion and fission of the multi-layered epithelial mass. Finally, our model also allowed epithelial cell polarisation and follicular lumen formation by endothelial cell abundance and proximity. Our study illustrates how constant discussion between theoretical and experimental approaches can help us to better understand the roles of cellular movement, adhesion and polarisation during thyroid embryonic development. We anticipate that the use of in silico models like the one we describe can push forward the fields of developmental biology and regenerative medicine.