Microvilli-derived Extracellular Vesicles Govern Morphogenesis in Drosophila wing epithelium

ABSTRACTThe regulation and coordination of developmental processes involves the secretion of morphogens and membrane carriers, including extracellular vesicles, which facilitate their transport over long distance. The long-range activity of the Hedgehog morphogen is conveyed by extracellular vesicles. However, the site and the molecular basis of their biogenesis remains unknown. By combining fluorescence and electron microscopy combined with genetics and cell biology approaches, we investigated the origin and the cellular mechanisms underlying extracellular vesicle biogenesis, and their contribution to Drosophila wing disc development, exploiting Hedgehog as a long-range morphogen. We show that microvilli of Drosophila wing disc epithelium are the site of generation of small extracellular vesicles that transport Hedgehog across the tissue. This process requires the Prominin-like protein, whose activity, together with interacting cytoskeleton components and lipids, is critical for maintaining microvilli integrity and function in secretion. Our results provide the first evidence that microvilli-derived extracellular vesicles contribute to Hedgehog long-range signaling activity highlighting their physiological significance in tissue development in vivo.

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Extracellular vesicles as mediators and markers of acute organ injury: current concepts

European Journal of Trauma and Emergency Surgery ◽

10.1007/s00068-021-01607-1 ◽

2021 ◽

Author(s):

Birte Weber ◽

Niklas Franz ◽

Ingo Marzi ◽

Dirk Henrich ◽

Liudmila Leppik

Keyword(s):

Extracellular Vesicles ◽

Inflammatory Diseases ◽

Organ Injury ◽

Isolation And Characterization ◽

Communication Processes ◽

Incidence And Mortality ◽

New Biomarkers ◽

And Function

AbstractDue to the continued high incidence and mortality rate worldwide, there is a need to develop new strategies for the quick, precise, and valuable recognition of presenting injury pattern in traumatized and poly-traumatized patients. Extracellular vesicles (EVs) have been shown to facilitate intercellular communication processes between cells in close proximity as well as distant cells in healthy and disease organisms. miRNAs and proteins transferred by EVs play biological roles in maintaining normal organ structure and function under physiological conditions. In pathological conditions, EVs change the miRNAs and protein cargo composition, mediating or suppressing the injury consequences. Therefore, incorporating EVs with their unique protein and miRNAs signature into the list of promising new biomarkers is a logical next step. In this review, we discuss the general characteristics and technical aspects of EVs isolation and characterization. We discuss results of recent in vitro, in vivo, and patients study describing the role of EVs in different inflammatory diseases and traumatic organ injuries. miRNAs and protein signature of EVs found in patients with acute organ injury are also debated.

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Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice

Biology ◽

10.3390/biology10010029 ◽

2021 ◽

Vol 10 (1) ◽

pp. 29

Author(s):

Raghubendra Singh Dagur ◽

Moses New-Aaron ◽

Murali Ganesan ◽

Weimin Wang ◽

Svetlana Romanova ◽

...

Keyword(s):

Oxidative Stress ◽

Extracellular Vesicles ◽

Treated Group ◽

Human Hepatocytes ◽

In Vivo Studies ◽

People Living With Hiv ◽

Humanized Mouse Model ◽

And Function

Background: Alcohol abuse is common in people living with HIV-1 and dramaticallyenhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomaldysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles(EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction. Methods:The study was performed on primary human hepatocytes and human hepatoma RLWXP-GFP (Huh7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liverhumanizedfumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chainknockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA virus. Results: We observedan increase in the secretion of EVs associated with a decrease in lysosomal activity and expressionof lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary humanhepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulatedgenes in the alcohol–HIV-treated group. Upstream regulator analysis of differentially expressedgenes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstreamgenes associated with increased oxidative stress, lysosomal associated disease, and function andEVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocytetransplantedhumanized mice, indicating that intensive EVs’ generation by human hepatocytes andtheir secretion to serum was associated with increased oxidative stress and reduction in lysosomalactivities triggered by HIV infection and ethanol diet. Conclusion: HIV-and-ethanol-metabolisminducedEVs release is tightly controlled by lysosome status in hepatocytes and participates in thedevelopment of double-insult-induced liver injury.

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STEM-18. STROMAL EXTRACELLULAR VESICLES DRIVE GLIOMA STEM CELL REPROGRAMMING

Neuro-Oncology ◽

10.1093/neuonc/noab196.092 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi24-vi25

Author(s):

Lata Adnani ◽

Brian Meehan ◽

Jordan Kassouf ◽

Cristiana Spinelli ◽

Nadim Tawil ◽

...

Keyword(s):

Molecular Subtype ◽

Extracellular Vesicle ◽

Host Cells ◽

Tumour Mass ◽

Membrane Structures ◽

And Function ◽

Rate Limiting ◽

The Brain

Abstract Glioblastoma multiforme (GBM) represents the most frequent and lethal form of brain tumors originating from glioma stem cells (GSCs). GBM remains lethal because the rate limiting patho-mechanisms remain poorly understood. In this regard, few limitations involve the diversity 'between' cellular states and the molecular/cellular complexity 'within' the tumour mass. Using cell based- and mouse- models, we explored extracellular vesicle (EV) mediated interactions between cancer and stromal cells impacting phenotypes of GSCs as a function of their molecular subtype. EVs are spherical membrane structures that cells release to expel different molecular cargo (lipids, proteins, RNA, DNA), which can be transported across a distance in the brain and taken up by various ‘recipient’ cells resulting in reprogramming of the recipient cell's content and function. In vivo, GSCs altered their pattern of NOTCH signalling and molecular phenotype as a function of proximity to non-transformed host cells in the brain. In vitro stromal EVs altered GSC sphere forming capacity, proteome and expression of mesenchymal markers. Thus, EV mediated tumour-stromal interactions could represent a biological switch and a novel targeting point in the biology of GBM.

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Application of Echocardiography on Transgenic Mice with Cardiomyopathies

Biochemistry Research International ◽

10.1155/2012/715197 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 6

Author(s):

G. Chen ◽

Y. Li ◽

J. Tian ◽

L. Zhang ◽

P. Jean-Charles ◽

...

Keyword(s):

Heart Failure ◽

Transgenic Mice ◽

Tissue Doppler ◽

Doppler Imaging ◽

Cellular Mechanisms ◽

Embryonic Mouse ◽

Experimental Animals ◽

Therapeutic Outcomes ◽

And Function

Cardiomyopathies are common cardiac disorders that primarily affect cardiac muscle resulting in cardiac dysfunction and heart failure. Transgenic mouse disease models have been developed to investigate the cellular mechanisms underlying heart failure and sudden cardiac death observed in cardiomyopathy cases and to explore the therapeutic outcomes in experimental animals in vivo. Echocardiography is an essential diagnostic tool for accurate and noninvasive assessment of cardiac structure and function in experimental animals. Our laboratory has been among the first to apply high-frequency research echocardiography on transgenic mice with cardiomyopathies. In this work, we have summarized our and other studies on assessment of systolic and diastolic dysfunction using conventional echocardiography, pulsed Doppler, and tissue Doppler imaging in transgenic mice with various cardiomyopathies. Estimation of embryonic mouse hearts has been performed as well using this high-resolution echocardiography. Some technical considerations in mouse echocardiography have also been discussed.

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Molecular mechanisms of olfactory detection in insects: beyond receptors

Open Biology ◽

10.1098/rsob.200252 ◽

2020 ◽

Vol 10 (10) ◽

pp. 200252

Author(s):

Hayden R. Schmidt ◽

Richard Benton

Keyword(s):

Molecular Mechanisms ◽

Olfactory Receptors ◽

Environmental Chemicals ◽

Ecological Niches ◽

Cellular Mechanisms ◽

Signalling Molecules ◽

Olfactory Systems ◽

Lymph Fluid ◽

And Function

Insects thrive in diverse ecological niches in large part because of their highly sophisticated olfactory systems. Over the last two decades, a major focus in the study of insect olfaction has been on the role of olfactory receptors in mediating neuronal responses to environmental chemicals. In vivo , these receptors operate in specialized structures, called sensilla, which comprise neurons and non-neuronal support cells, extracellular lymph fluid and a precisely shaped cuticle. While sensilla are inherent to odour sensing in insects, we are only just beginning to understand their construction and function. Here, we review recent work that illuminates how odour-evoked neuronal activity is impacted by sensillar morphology, lymph fluid biochemistry, accessory signalling molecules in neurons and the physiological crosstalk between sensillar cells. These advances reveal multi-layered molecular and cellular mechanisms that determine the selectivity, sensitivity and dynamic modulation of odour-evoked responses in insects.

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Exosomes and extracellular vesicles: the path forward

Essays in Biochemistry ◽

10.1042/ebc20170088 ◽

2018 ◽

Vol 62 (2) ◽

pp. 119-124 ◽

Cited By ~ 38

Author(s):

Philip D. Stahl ◽

Graça Raposo

Keyword(s):

Extracellular Vesicles ◽

Cell Biology ◽

Biological Fluids ◽

Therapeutic Agents ◽

Tissue Homeostasis ◽

The Past ◽

And Function ◽

Intercellular Communications ◽

Biomedical Community

Over the course of the past several decades, the concept that extracellular vesicles, exosomes and microvesicles, operate as cellular “housekeepers” and as agents for communication between and among cells and tissues, has emerged into one of the most promising yet vexing problems facing the biomedical community. Already, extracellular vesicles from biological fluids are being used for diagnostic purposes and hopes abound for their use as therapeutic agents. However, the most basic mechanistic questions surrounding their biogenesis and function in cellular and tissue homeostasis remain largely unexplored. In this issue of Essays in Biochemistry, the rise of a new intercellular communications pathway is considered from many perspectives—cell biology, physiology, and pathophysiology.

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Mesenchymal Stem Cell-Derived Extracellular Vesicles Improve the Renal Microvasculature in Metabolic Renovascular Disease in Swine

Cell Transplantation ◽

10.1177/0963689718780942 ◽

2018 ◽

Vol 27 (7) ◽

pp. 1080-1095 ◽

Cited By ~ 31

Author(s):

Alfonso Eirin ◽

Xiang-Yang Zhu ◽

Sreela Jonnada ◽

Amir Lerman ◽

Andre J. van Wijnen ◽

...

Keyword(s):

Extracellular Vesicles ◽

Therapeutic Potential ◽

Ex Vivo ◽

Capillary Density ◽

Swine Model ◽

Renovascular Disease ◽

And Function ◽

Angiogenic Genes ◽

Endothelial Growth Factor

Background: Extracellular vesicles (EVs) released from mesenchymal stem/stromal cells (MSCs) mediate their paracrine effect, but their efficacy to protect the microcirculation of the kidney is unknown. Using a novel swine model of unilateral renovascular disease (RVD) complicated by metabolic syndrome (MetS), we tested the hypothesis that EVs would attenuate renal microvascular loss. Methods: Four groups of pigs ( n = 7 each) were studied after 16 weeks of diet-induced MetS and RVD (MetS+RVD), MetS+RVD treated 4 weeks earlier with a single intra-renal delivery of EVs harvested from autologous adipose tissue-derived MSCs, and Lean and MetS Sham controls. Stenotic-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were measured in-vivo (fast CT), whereas EV characteristics, renal microvascular architecture (micro-CT), and injury pathways were studied ex-vivo. Results: mRNA sequencing and proteomic analysis revealed that EVs are packed with several pro-angiogenic genes and proteins, such as vascular endothelial growth factor. Labeled EVs were detected in the stenotic kidney 4 weeks after injection internalized by tubular and endothelial cells. EVs restored renal expression of angiogenic factors and improved cortical microvascular and peritubular capillary density. Renal apoptosis, oxidative stress, tubular injury, and fibrosis were also attenuated in EV-treated pigs. RBF and GFR decreased in MetS+RVD compared with MetS, but normalized in MetS+RVD+EVs. Conclusions: Intra-renal delivery of MSC-derived EVs bearing pro-angiogenic properties restored the renal microcirculation and in turn hemodynamics and function in chronic experimental MetS+RVD. Our study suggests a novel therapeutic potential for MSC-derived EVs in restoring renal hemodynamics in experimental MetS+RVD.

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An Assay to Detect In Vivo Y Chromosome Loss in Drosophila Wing Disc Cells

G3 Genes|Genome|Genetics ◽

10.1534/g3.112.002899 ◽

2012 ◽

Vol 2 (9) ◽

pp. 1095-1102 ◽

Cited By ~ 9

Author(s):

Janos Szabad ◽

Hugo J. Bellen ◽

Koen J. T. Venken

Keyword(s):

Y Chromosome ◽

Wing Disc ◽

Chromosome Loss ◽

Drosophila Wing ◽

Disc Cells

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Kruppel-Like Factors in Thrombosis and Hemostasis

Blood ◽

10.1182/blood-2018-99-109558 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. SCI-45-SCI-45

Author(s):

Mukesh Jain

Keyword(s):

Cell Biology ◽

Vascular System ◽

Conflicts Of Interest ◽

Human Subjects ◽

Myeloid Cell ◽

Nutrient Delivery ◽

Inflammatory Processes ◽

Endothelial Functions ◽

And Function

Abstract Armed with the appreciation that the blood and vascular system share common origins and cooperate to ensure fundamental processes (e.g. blood flow/fluidity, oxygen/nutrient delivery, immunity) essential for organismal survival, we posited that shared molecular pathways may be operative in coordinating the function of both systems. Over the past 2 decades, studies from our group and others have identified a family of transcription factors termed Kruppel-like factors (KLFs) as essential for development, differentiation, and function of cellular constituents of both the hematopoietic and vascular systems. In this presentation, discussion will focus on the role KLFs in control of endothelium and myeloid cell biology in physiology and disease. Specifically, cellular and in vivo evidence will be discussed implicating KLFs as master regulators of all cardinal endothelial functions (permeability, vasoreactivity, blood fluidity, and inflammation). Further, studies demonstrating KLF-control of myeloid cell development, subset specification, and pro-inflammatory activation will be reviewed with particular emphasis on results of efforts altering myeloid KLFs in the context of acute (e.g. bacterial infection, sepsis) and chronic (e.g. atherosclerosis, arterial/venous thrombosis) inflammatory processes. Correlative studies in human subjects will be presented. And finally, insights into how targeting KLFs can be exploited for therapeutic gain will be discussed. Disclosures No relevant conflicts of interest to declare.

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Skeletal muscle releases extracellular vesicles with distinct protein and miRNA signatures that accumulate and function within the muscle microenvironment

10.1101/2021.11.30.470551 ◽

2021 ◽

Author(s):

Sho Watanabe ◽

Yuri Sudo ◽

Satoshi Kimura ◽

Kenji Tomita ◽

Makoto Noguchi ◽

...

Keyword(s):

Skeletal Muscle ◽

Extracellular Vesicles ◽

Cell Types ◽

The Body ◽

C2c12 Cells ◽

Ips Cell ◽

Potential Marker ◽

And Function ◽

Intercellular Communications

Extracellular vesicles (EVs) contain various regulatory molecules and mediate intercellular communications. Although EVs are secreted from various cell types, including skeletal muscle cells, and present in the blood, their identity is poorly characterized in vivo, limiting the identification of their origin in the blood. Since the skeletal muscle is the largest organ in the body, it could substantially contribute to circulating EVs as their source. However, due to the lack of defined markers that distinguish SkM-EVs from others, whether the skeletal muscle releases EVs in vivo and how much the skeletal muscle-derived EVs (SkM-EVs) account for plasma EVs remain poorly understood. In this work, we perform quantitative proteomic analyses on EVs released from C2C12 cells and human iPS cell-derived myocytes and identify potential marker proteins that mark SkM-EVs. These markers we identified apply to in vivo tracking of SkM-EVs. The results show that skeletal muscle makes only a subtle contribution to plasma EVs as their source in both control and exercise conditions in mice. On the other hand, we demonstrate that SkM-EVs are concentrated in the skeletal muscle interstitium. Furthermore, we show that interstitium EVs are highly enriched with the muscle-specific miRNAs and repress the expression of the paired box transcription factor Pax7, a master regulator for myogenesis. Taken together, our findings reveal that the skeletal muscle releases exosome-like small EVs with distinct protein and miRNA profiles in vivo and that SkM-EVs mainly play a role within the muscle microenvironment where they accumulate.

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