Publications by authors named "Bas W M van Balkom"

47 Publications

Functional assays to assess the therapeutic potential of extracellular vesicles.

J Extracell Vesicles 2020 Nov 29;10(1):e12033. Epub 2020 Nov 29.

Department of Nephrology and Hypertension UMC Utrecht Utrecht The Netherlands.

An important aspect in the development of extracellular vesicle (EV) therapeutics is identifying and quantifying the key features defining their identity, purity, sterility, potency and stability to ensure batch-to-batch reproducibility of their therapeutic efficacy. Apart from EV-inherent features, therapeutic efficacy depends on a variety of additional parameters, like dosing, frequency of application, and administration route, some of which can be addressed only in clinical trials. Before initiating clinical trials, EV-inherent features should be tested in well-standardized quantitative assays or in appropriate animal models . Ideally, such assays would predict if a particular EV preparation has the potential to achieve its intended therapeutic effects, and could be further developed into formal potency assays as published by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guidelines. Furthermore, such assays should facilitate the comparison of EV preparations produced in different batches, on different manufacturing platforms or deriving from different cell sources. For now, a wide spectrum of and assays has been used to interrogate the therapeutic functions of EVs. However, many cannot accurately predict therapeutic potential. Indeed, several unique challenges make it difficult to set up reliable assays to assess the therapeutic potential of EVs, and to develop such assays into formal potency tests. Here, we discuss challenges and opportunities around and testing of EV therapeutic potential, including the need for harmonization, establishment of formal potency assays and novel developments for functional testing.
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http://dx.doi.org/10.1002/jev2.12033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890556PMC
November 2020

A new microfluidic model that allows monitoring of complex vascular structures and cell interactions in a 3D biological matrix.

Lab Chip 2020 05;20(10):1827-1844

Department of Nephrology and Hypertension, University Medical Center Utrecht, PO Box 85500, 3584 CX Utrecht, The Netherlands.

Microfluidic organ-on-a-chip designs are used to mimic human tissues, including the vasculature. Here we present a novel microfluidic device that allows the interaction of endothelial cells (ECs) with pericytes and the extracellular matrix (ECM) in full bio-matrix encased 3D vessel structures (neovessels) that can be subjected to continuous, unidirectional flow and perfusion with circulating immune cells. We designed a polydimethylsiloxane (PDMS) device with a reservoir for a 3D fibrinogen gel with pericytes. Open channels were created for ECs to form a monolayer. Controlled, continuous, and unidirectional flow was introduced via a pump system while the design facilitated 3D confocal imaging. In this vessel-on-a-chip system, ECs interact with pericytes to create a human cell derived blood vessel which maintains a perfusable lumen for up to 7 days. Dextran diffusion verified endothelial barrier function while demonstrating the beneficial role of supporting pericytes. Increased permeability after thrombin stimulation showed the capacity of the neovessels to show natural vascular response. Perfusion of neovessels with circulating THP-1 cells demonstrated this system as a valuable platform for assessing interaction between the endothelium and immune cells in response to TNFα. In conclusion: we created a novel vascular microfluidic device that facilitates the fabrication of an array of parallel soft-channel structures in ECM gel that develop into biologically functional neovessels without hard-scaffold support. This model provides a unique tool to conduct live in vitro imaging of the human vasculature during perfusion with circulating cells to mimic (disease) environments in a highly systematic but freely configurable manner.
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http://dx.doi.org/10.1039/d0lc00059kDOI Listing
May 2020

Paracrine Proangiogenic Function of Human Bone Marrow-Derived Mesenchymal Stem Cells Is Not Affected by Chronic Kidney Disease.

Stem Cells Int 2019 23;2019:1232810. Epub 2019 Dec 23.

Department of Nephrology and Hypertension, Regenerative Medicine Center Utrecht, UMC Utrecht, Utrecht University, Uppsalalaan 8, 3584 CT, Utrecht, Netherlands.

Background: Cell-based therapies are being developed to meet the need for curative therapy in chronic kidney disease (CKD). Bone marrow- (BM-) derived mesenchymal stromal cells (MSCs) enhance tissue repair and induce neoangiogenesis through paracrine action of secreted proteins and extracellular vesicles (EVs). Administration of allogeneic BM MSCs is less desirable in a patient population likely to require a kidney transplant, but potency of autologous MSCs should be confirmed, given previous indications that CKD-induced dysfunction is present. While the immunomodulatory capacity of CKD BM MSCs has been established, it is unknown whether CKD affects wound healing and angiogenic potential of MSC-derived CM and EVs.

Methods: MSCs were cultured from BM obtained from kidney transplant recipients ( = 15) or kidney donors ( = 17). Passage 3 BM MSCs and BM MSC-conditioned medium (CM) were used for experiments. EVs were isolated from CM by differential ultracentrifugation. BM MSC differentiation capacity, proliferation, and senescence-associated -galactosidase activity was assessed. promigratory and proangiogenic capacity of BM MSC-derived CM and EVs was assessed using an scratch wound assay and Matrigel angiogenesis assay.

Results: Healthy and CKD BM MSCs exhibited similar differentiation capacity, proliferation, and senescence-associated -galactosidase activity. Scratch wound migration was not significantly different between healthy and CKD MSCs ( = 0.18). Healthy and CKD BM MSC-derived CM induced similar tubule formation ( = 0.21). There was also no difference in paracrine regenerative function of EVs (scratch wound: = 0.6; tubulogenesis: = 0.46).

Conclusions: Our results indicate that MSCs have an intrinsic capacity to produce proangiogenic paracrine factors, including EVs, which is not affected by donor health status regarding CKD. This suggests that autologous MSC-based therapy is a viable option in CKD.
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http://dx.doi.org/10.1155/2019/1232810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942892PMC
December 2019

The Small RNA Repertoire of Small Extracellular Vesicles Isolated From Donor Kidney Preservation Fluid Provides a Source for Biomarker Discovery for Organ Quality and Posttransplantation Graft Function.

Transplant Direct 2019 Sep 12;5(9):e484. Epub 2019 Aug 12.

Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.

Delayed graft function (DGF) after kidney transplantation is negatively associated with long-term graft function and survival. Kidney function after transplantation depends on multiple factors, both donor- and recipient-associated. Prediction of posttransplantation graft function would allow timely intervention to optimize patient care and survival. Currently, graft-based predictions can be made based on histological and molecular analyses of 0-hour biopsy samples. However, such analyses are currently not implemented, as biopsy samples represent only a very small portion of the entire graft and are not routinely analyzed in all transplantation centers. Alternatives are thus required.

Methods: We analyzed whether donor organ preservation fluid contain small extracellular vesicles (sEV) and whether the RNA content of these vesicles could be used as a source for potential biomarkers for posttransplantation kidney function.

Results: We provide proof of principle that sEVs are present in preservation fluid, which contain RNAs associated with donor origin. Furthermore, sEV micro RNA profiles could be associated with graft function during the first 7 days posttransplantation, but no significant correlation with DGF could be established based on the current dataset.

Conclusions: Overall, the predictive potential of sEV RNA biomarkers together with relatively easy and noninvasive sample collection and analysis methods could pave the way towards universal screening of donor kidney-associated risk for DGF, optimized patient treatment, and subsequently improved short- and long-term graft function and survival.
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http://dx.doi.org/10.1097/TXD.0000000000000929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739040PMC
September 2019

Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications.

J Extracell Vesicles 2019 29;8(1):1609206. Epub 2019 Apr 29.

Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore.

Small extracellular vesicles (sEVs) from mesenchymal stromal/stem cells (MSCs) are transiting rapidly towards clinical applications. However, discrepancies and controversies about the biology, functions, and potency of MSC-sEVs have arisen due to several factors: the diversity of MSCs and their preparation; various methods of sEV production and separation; a lack of standardized quality assurance assays; and limited reproducibility of and functional assays. To address these issues, members of four societies (SOCRATES, ISEV, ISCT and ISBT) propose specific harmonization criteria for MSC-sEVs to facilitate data sharing and comparison, which should help to advance the field towards clinical applications. Specifically, MSC-sEVs should be defined by quantifiable metrics to identify the cellular origin of the sEVs in a preparation, presence of lipid-membrane vesicles, and the degree of physical and biochemical integrity of the vesicles. For practical purposes, new MSC-sEV preparations might also be measured against a well-characterized MSC-sEV biological reference. The ultimate goal of developing these metrics is to map aspects of MSC-sEV biology and therapeutic potency onto quantifiable features of each preparation.
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http://dx.doi.org/10.1080/20013078.2019.1609206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6493293PMC
April 2019

Characterization of Endothelial and Smooth Muscle Cells From Different Canine Vessels.

Front Physiol 2019 12;10:101. Epub 2019 Feb 12.

Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.

Vasculature performs a critical function in tissue homeostasis, supply of oxygen and nutrients, and the removal of metabolic waste products. Vascular problems are implicated in a large variety of pathologies and accurate models resembling native vasculature are of great importance. Unfortunately, existing models do not sufficiently reflect their counterpart. The complexity of vasculature requires the examination of multiple cell types including endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), as well as vessel location in the body from which they originate. The use of canine blood vessels provides a way to study vasculature with similar vessel size and physiology compared to human vasculature. We report an isolation procedure that provides the possibility to isolate both the endothelial and smooth muscle cells from the same vessels simultaneously, enabling new opportunities in investigating vasculature behavior. Canine primary ECs and VSMCs were isolated from the vena cava, vena porta and aorta. All tissue sources were derived from three donors for accurate comparison and to reduce inter-animal variation. The isolation and purification of the two distinct cell types was confirmed by morphology, gene- and protein-expression and function. As both cell types can be derived from the same vessel, this approach allows accurate modeling of vascular diseases and can also be used more widely, for example, in vascular bioreactors and tissue engineering designs. Additionally, we identified several new genes that were highly expressed in canine ECs, which may become candidate genes for novel EC markers. In addition, we observed transcriptional and functional differences between arterial- and venous-derived endothelium. Further exploration of the transcriptome and physiology of arteriovenous differentiation of primary cells may have important implications for a better understanding of the fundamental behavior of the vasculature and pathogenesis of vascular disease.
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http://dx.doi.org/10.3389/fphys.2019.00101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379353PMC
February 2019

Proteomic Signature of Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles.

Proteomics 2019 01 4;19(1-2):e1800163. Epub 2019 Jan 4.

Institute of Medical Biology, Agency for Science Technology and Research (A*STAR), 138648, Singapore.

Small extracellular vesicles (EVs) are 50-200 nm vesicles secreted by most cells. They are considered as mediators of intercellular communication, and EVs from specific cell types, in particular mesenchymal stem/stromal cells (MSCs), offer powerful therapeutic potential, and can provide a novel therapeutic strategy. They appear promising and safe (as EVs are non-self-replicating), and eventually MSC-derived EVs (MSC-EVs) may be developed to standardized, off-the-shelf allogeneic regenerative and immunomodulatory therapeutics. Promising pre-clinical data have been achieved using MSCs from different sources as EV-producing cells. Similarly, a variety EV isolation and characterization methods have been applied. Interestingly, MSC-EVs obtained from different sources and prepared with different methods show in vitro and in vivo therapeutic effects, indicating that isolated EVs share a common potential. Here, well-characterized and controlled, publicly available proteome profiles of MSC-EVs are compared to identify a common MSC-EV protein signature that might be coupled to the MSC-EVs' common therapeutic potential. This protein signature may be helpful in developing MSC-EV quality control platforms required to confirm the identity and test for the purity of potential therapeutic MSC-EVs.
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http://dx.doi.org/10.1002/pmic.201800163DOI Listing
January 2019

Lysyl oxidase-like 2 is a regulator of angiogenesis through modulation of endothelial-to-mesenchymal transition.

J Cell Physiol 2019 07 1;234(7):10260-10269. Epub 2018 Nov 1.

Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

Lysyl oxidase-like 2 (LOXL2) belongs to the family of lysyl oxidases, and as such promotes crosslinking of collagens and elastin by oxidative deamination of lysine residues. In endothelial cells (ECs), LOXL2 is involved in crosslinking and scaffolding of collagen IV. Additionally, several reports have shown a role for LOXL2 in other processes, including regulation of gene expression, tumor metastasis, and epithelial-to-mesenchymal transition (EMT). Here, we demonstrate an additional role for LOXL2 in the regulation of angiogenesis by modulation of endothelial-to-mesenchymal transition (EndMT). LOXL2 knockdown in ECs results in decreased migration and sprouting, and concordantly, LOXL2 overexpression leads to an increase in migration and sprouting, independent of its catalytic activity. Furthermore, LOXL2 knockdown resulted in a reduced expression of EndMT markers, and inhibition of transforming growth factor-β (TGF-β)-mediated induction of EndMT. Interestingly, unlike in EMT, overexpression of LOXL2 alone is insufficient to induce EndMT. Further investigation revealed that LOXL2 expression regulates protein kinase B (PKB)/Akt and focal adhesion kinase (FAK) signaling, both pathways that have been implicated in the regulation of EMT. Altogether, our studies reveal a role for LOXL2 in angiogenesis through the modulation of EndMT in ECs, independent of its enzymatic crosslinking activity.
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http://dx.doi.org/10.1002/jcp.27695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6587725PMC
July 2019

Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles.

Stem Cells Transl Med 2017 08 17;6(8):1730-1739. Epub 2017 Jul 17.

Institute of Medical Biology, A*STAR, Singapore.

Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell-related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV-based therapeutics at the preclinical and clinical levels. This review outlines topic-specific action items that, if addressed, will enhance the development of best-practice models for EV therapies. Stem Cells Translational Medicine 2017;6:1730-1739.
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http://dx.doi.org/10.1002/sctm.17-0055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689784PMC
August 2017

Proteins in Preservation Fluid as Predictors of Delayed Graft Function in Kidneys from Donors after Circulatory Death.

Clin J Am Soc Nephrol 2017 May;12(5):817-824

Departments of Nephrology and Hypertension and.

Background And Objectives: Kidney transplantation is the preferred treatment for ESRD, and donor kidney shortage urges proper donor-recipient matching. Zero-hour biopsies provide predictive values for short- and long-term transplantation outcomes, but are invasive and may not reflect the entire organ. Alternative, more representative methods to predict transplantation outcome are required. We hypothesized that proteins accumulating in preservation fluid during cold ischemic storage can serve as biomarkers to predict post-transplantation graft function.

Design, Setting, Participants, & Measurements: Levels of 158 proteins were measured in preservation fluids from kidneys donated after circulatory death (Maastricht category III) collected in two Dutch centers (University Medical Center Utrecht and Erasmus Medical Center Rotterdam) between 2013 and 2015. Five candidate biomarkers identified in a discovery set of eight kidneys with immediate function (IF) versus eight with delayed graft function (DGF) were subsequently analyzed in a verification set of 40 additional preservation fluids to establish a prediction model.

Results: Variables tested for their contribution to a prediction model included five proteins (leptin, periostin, GM-CSF, plasminogen activator inhibitor-1, and osteopontin) and two clinical parameters (recipient body mass index [BMI] and dialysis duration) that distinguished between IF and DGF in the discovery set. Stepwise multivariable logistic regression provided a prediction model on the basis of leptin and GM-CSF. Receiver operating characteristic analysis showed an area under the curve (AUC) of 0.87, and addition of recipient BMI generated a model with an AUC of 0.89, outperforming the Kidney Donor Risk Index and the DGF risk calculator, showing AUCs of 0.55 and 0.59, respectively.

Conclusions: We demonstrate that donor kidney preservation fluid harbors biomarkers that, together with information on recipient BMI, predict short-term post-transplantation kidney function. Our approach is safe, easy, and performs better than current prediction algorithms, which are only on the basis of clinical parameters.
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http://dx.doi.org/10.2215/CJN.10701016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477220PMC
May 2017

Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.

J Extracell Vesicles 2017 7;6(1):1286095. Epub 2017 Mar 7.

Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University , Utrecht , the Netherlands.

The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA . These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge - of the nature of EV(-RNA)s and of how to effectively and reliably study them - currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.
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http://dx.doi.org/10.1080/20013078.2017.1286095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345583PMC
March 2017

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.

J Extracell Vesicles 2015 31;4:30087. Epub 2015 Dec 31.

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany;

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4698466PMC
http://dx.doi.org/10.3402/jev.v4.30087DOI Listing
January 2016

Exosomes from hypoxic endothelial cells have increased collagen crosslinking activity through up-regulation of lysyl oxidase-like 2.

J Cell Mol Med 2016 Feb 27;20(2):342-50. Epub 2015 Nov 27.

Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.

Exosomes are important mediators of intercellular communication. Additionally, they contain a variety of components capable of interacting with the extracellular matrix (ECM), including integrins, matrix metalloproteinases and members of the immunoglobin superfamily. Despite these observations, research on exosome-ECM interactions is limited. Here, we investigate whether the exosome-associated lysyl oxidase family member lysyl oxidase-like 2 (LOXL2) is involved in ECM remodelling. We found that LOXL2 is present on the exterior of endothelial cell (EC)-derived exosomes, placing it in direct vicinity of the ECM. It is up-regulated twofold in EC-derived exosomes cultured under hypoxic conditions. Intact exosomes from hypoxic EC and LOXL2 overexpressing EC show increased activity in a fluorometric lysyl oxidase enzymatic activity assay as well as in a collagen gel contraction assay. Concordantly, knockdown of LOXL2 in exosome-producing EC in both normal and hypoxic conditions reduces activity of exosomes in both assays. Our findings show for the first time that ECM crosslinking by EC-derived exosomes is mediated by LOXL2 under the regulation of hypoxia, and implicate a role for exosomes in hypoxia-regulated focal ECM remodelling, a key process in both fibrosis and wound healing.
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http://dx.doi.org/10.1111/jcmm.12730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727569PMC
February 2016

Screen-based identification and validation of four new ion channels as regulators of renal ciliogenesis.

J Cell Sci 2015 Dec 6;128(24):4550-9. Epub 2015 Nov 6.

Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht 3584 CX, The Netherlands

To investigate the contribution of ion channels to ciliogenesis, we carried out a small interfering RNA (siRNA)-based reverse genetics screen of all ion channels in the mouse genome in murine inner medullary collecting duct kidney cells. This screen revealed four candidate ion channel genes: Kcnq1, Kcnj10, Kcnf1 and Clcn4. We show that these four ion channels localize to renal tubules, specifically to the base of primary cilia. We report that human KCNQ1 Long QT syndrome disease alleles regulate renal ciliogenesis; KCNQ1-p.R518X, -p.A178T and -p.K362R could not rescue ciliogenesis after Kcnq1-siRNA-mediated depletion in contrast to wild-type KCNQ1 and benign KCNQ1-p.R518Q, suggesting that the ion channel function of KCNQ1 regulates ciliogenesis. In contrast, we demonstrate that the ion channel function of KCNJ10 is independent of its effect on ciliogenesis. Our data suggest that these four ion channels regulate renal ciliogenesis through the periciliary diffusion barrier or the ciliary pocket, with potential implication as genetic contributors to ciliopathy pathophysiology. The new functional roles of a subset of ion channels provide new insights into the disease pathogenesis of channelopathies, which might suggest future therapeutic approaches.
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http://dx.doi.org/10.1242/jcs.176065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4696498PMC
December 2015

Quantitative and qualitative analysis of small RNAs in human endothelial cells and exosomes provides insights into localized RNA processing, degradation and sorting.

J Extracell Vesicles 2015 29;4:26760. Epub 2015 May 29.

Department of Nephrology and Hypertension, UMC Utrecht, Utrecht, the Netherlands.

Exosomes are small vesicles that mediate cell-cell communication. They contain proteins, lipids and RNA, and evidence is accumulating that these molecules are specifically sorted for release via exosomes. We recently showed that endothelial-cell-produced exosomes promote angiogenesis in vivo in a small RNA-dependent manner. Recent deep sequencing studies in exosomes from lymphocytic origin revealed a broad spectrum of small RNAs. However, selective depletion or incorporation of small RNA species into endothelial exosomes has not been studied extensively. With next generation sequencing, we identified all known non-coding RNA classes, including microRNAs (miRNAs), small nucleolar RNAs, yRNAs, vault RNAs, 5p and 3p fragments of miRNAs and miRNA-like fragments. In addition, we mapped many fragments of messenger RNAs (mRNAs) and mitochondrial RNAs (mtRNAs). The distribution of small RNAs in exosomes revealed a considerable overlap with the distribution in the producing cells. However, we identified a remarkable enrichment of yRNA fragments and mRNA degradation products in exosomes consistent with yRNAs having a role in degradation of structured and misfolded RNAs in close proximity to endosomes. We propose that endothelial endosomes selectively sequester cytoplasmic RNA-degrading machineries taking part in gene regulation. The release of these regulatory RNAs via exosomes may have implications for endothelial cell-cell communication.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450249PMC
http://dx.doi.org/10.3402/jev.v4.26760DOI Listing
June 2015

Extracellular vesicles: potential roles in regenerative medicine.

Front Immunol 2014 3;5:608. Epub 2014 Dec 3.

Department of Nephrology and Hypertension, University Medical Center Utrecht , Utrecht , Netherlands.

Extracellular vesicles (EV) consist of exosomes, which are released upon fusion of the multivesicular body with the cell membrane, and microvesicles, which are released directly from the cell membrane. EV can mediate cell-cell communication and are involved in many processes, including immune signaling, angiogenesis, stress response, senescence, proliferation, and cell differentiation. The vast amount of processes that EV are involved in and the versatility of manner in which they can influence the behavior of recipient cells make EV an interesting source for both therapeutic and diagnostic applications. Successes in the fields of tumor biology and immunology sparked the exploration of the potential of EV in the field of regenerative medicine. Indeed, EV are involved in restoring tissue and organ damage, and may partially explain the paracrine effects observed in stem cell-based therapeutic approaches. The function and content of EV may also harbor information that can be used in tissue engineering, in which paracrine signaling is employed to modulate cell recruitment, differentiation, and proliferation. In this review, we discuss the function and role of EV in regenerative medicine and elaborate on potential applications in tissue engineering.
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http://dx.doi.org/10.3389/fimmu.2014.00608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4253973PMC
December 2014

EVpedia: a community web portal for extracellular vesicles research.

Bioinformatics 2015 Mar 10;31(6):933-9. Epub 2014 Nov 10.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea, Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Republic of Korea, Cardiovascular Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA, Department of Clinical Immunology, Polish-American Institute of Paediatrics, Jagiellonian University Medical College, Cracow, Poland, Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands, Section of Oncology, Department of Clinical Sciences, Lund University, Lund, Sweden, The Feinstein Institute for Medical Research, Manhasset, NY, USA, Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, Atlanta, GA, USA, Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council (CNR), Palermo, Italy, Innovation in Vesicles and Cells for Application in Therapy, Germans Trias i Pujol Research Institute, Germans Trias i Pujol University Hospital, Badalona, Spain, INSERM, UMR837 JEAN-PIERRE Aubert Research Centre, Lille, France, Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary, Department of Biochemistry and Molecular Biology, BIO21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC, Australia, Institute of Cancer & Genetics, School of Medicine, Velindre Cancer Centre, Cardiff University, Cardiff, UK, Program in Cellular and Molecular Medicine at Boston Children's Hospital and Department of Cell Biology, Harvard Medical School, Boston, MA, USA, Section of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA, Department of Neurology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, USA, Cancer Biology Program, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai M

Motivation: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging.

Results: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research.

Availability And Implementation: The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.
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http://dx.doi.org/10.1093/bioinformatics/btu741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375401PMC
March 2015

Quantitative proteomic identification of host factors involved in the Salmonella typhimurium infection cycle.

Methods Mol Biol 2015 ;1225:29-45

Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM, Utrecht, The Netherlands.

Quantitative proteomics, based on stable isotope labeling by amino acids in cell culture (SILAC), can be used to identify host proteins involved in the intracellular interplay with pathogens. This method allows identification of proteins subject to degradation or upregulation in response to intracellular infection. It can also be used to study intracellular dynamics (trafficking) of proteins in response to the infection. Here, we describe the analysis of changes in protein profiles determined in Golgi-enriched fractions isolated from cells that were either mock-infected or infected with Salmonella typhimurium. Using the SILAC approach we were able to identify 105 proteins in Golgi-enriched fractions that were significantly changed in their abundance as a result of Salmonella infection.
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http://dx.doi.org/10.1007/978-1-4939-1625-2_2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7121999PMC
May 2016

The potential of exosomes in diagnosis and treatment of inborn errors of metabolism.

J Inherit Metab Dis 2014 Jul 8;37(4):497-504. Epub 2014 Feb 8.

Department of Nephrology and Hypertension, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands,

Extracellular vesicles, in particular exosomes, have gained much attention as potent mediators of intercellular signaling. Exosomes are 50-130 nm intraluminal vesicles of multivesicular bodies (MVB) that are secreted into the extracellular environment upon fusion of MVB with the plasma membrane. Current research on exosomes focuses on their biogenesis, including specific sorting mechanisms, their potential to transfer proteins and RNA from their cells of origin to target cells, specific methods of vesicle isolation, and their possible application as diagnostic and therapeutic devices. Exosomes are vesicles of endocytic origin that contain a portion of the cytoplasm. Their molecular components represent the composition and thereby the physiological state of the cells from which they originate. In this review, we recapitulate the discovery of exosomes and the subsequent expansion of exosome research into a variety of different areas of interest, with a specific focus on how exosomes could prove to be invaluable for both diagnostic and therapeutic applications within the research field of inborn errors of metabolism.
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http://dx.doi.org/10.1007/s10545-014-9681-zDOI Listing
July 2014

Human adipocyte extracellular vesicles in reciprocal signaling between adipocytes and macrophages.

Obesity (Silver Spring) 2014 May 9;22(5):1296-308. Epub 2014 Jan 9.

Department of Vascular Medicine, University Medical Center Utrecht (UMC Utrecht), Utrecht, The Netherlands; Section Metabolic Diseases, Molecular Cancer Research, UMC Utrecht, Utrecht, The Netherlands.

Objective: Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)-explants play a role in the paracrine interaction between adipocytes and macrophages, a key mechanism in AT inflammation, leading to metabolic complications like insulin resistance (IR) were determined.

Methods: EVs released from in vitro differentiated adipocytes and AT-explants ex vivo were characterized by electron microscopy, Western blot, multiplex adipokine-profiling, and quantified by flow cytometry. Primary monocytes were stimulated with EVs from adipocytes, subcutaneous (SCAT) or omental-derived AT (OAT), and phenotyped. Macrophage supernatant was subsequently used to assess the effect on insulin signaling in adipocytes.

Results: Adipocyte and AT-derived EVs differentiated monocytes into macrophages characteristic of human adipose tissue macrophages (ATM), defined by release of both pro- and anti-inflammatory cytokines. The adiponectin-positive subset of AT-derived EVs, presumably representing adipocyte-derived EVs, induced a more pronounced ATM-phenotype than the adiponectin-negative AT-EVs. This effect was more evident for OAT-EVs versus SCAT-EVs. Furthermore, supernatant of macrophages pre-stimulated with AT-EVs interfered with insulin signaling in human adipocytes. Finally, the number of OAT-derived EVs correlated positively with patients HOMA-IR.

Conclusions: A possible role for human AT-EVs in a reciprocal pro-inflammatory loop between adipocytes and macrophages, with the potential to aggravate local and systemic IR was demonstrated.
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http://dx.doi.org/10.1002/oby.20679DOI Listing
May 2014

Endothelial cells require miR-214 to secrete exosomes that suppress senescence and induce angiogenesis in human and mouse endothelial cells.

Blood 2013 May 26;121(19):3997-4006, S1-15. Epub 2013 Mar 26.

Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.

Signaling between endothelial cells, endothelial progenitor cells, and stromal cells is crucial for the establishment and maintenance of vascular integrity and involves exosomes, among other signaling pathways. Exosomes are important mediators of intercellular communication in immune signaling, tumor survival, stress responses, and angiogenesis. The ability of exosomes to incorporate and transfer messenger RNAs (mRNAs) encoding for "acquired" proteins or micro RNAs (miRNAs) repressing "resident" mRNA translation suggests that they can influence the physiological behavior of recipient cells. We demonstrate that miR-214, an miRNA that controls endothelial cell function and angiogenesis, plays a dominant role in exosome-mediated signaling between endothelial cells. Endothelial cell-derived exosomes stimulated migration and angiogenesis in recipient cells, whereas exosomes from miR-214-depleted endothelial cells failed to stimulate these processes. Exosomes containing miR-214 repressed the expression of ataxia telangiectasia mutated in recipient cells, thereby preventing senescence and allowing blood vessel formation. Concordantly, specific reduction of miR-214 content in exosome-producing endothelial cells abolishes the angiogenesis stimulatory function of the resulting exosomes. Collectively, our data indicate that endothelial cells release miR-214-containing exosomes to stimulate angiogenesis through the silencing of ataxia telangiectasia mutated in neighboring target cells.
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http://dx.doi.org/10.1182/blood-2013-02-478925DOI Listing
May 2013

Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation.

PLoS Biol 2012 18;10(12):e1001450. Epub 2012 Dec 18.

Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.
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http://dx.doi.org/10.1371/journal.pbio.1001450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3525526PMC
May 2013

Human embryonic mesenchymal stem cell-derived conditioned medium rescues kidney function in rats with established chronic kidney disease.

PLoS One 2012 19;7(6):e38746. Epub 2012 Jun 19.

Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, the Netherlands.

Chronic kidney disease (CKD) is a major health care problem, affecting more than 35% of the elderly population worldwide. New interventions to slow or prevent disease progression are urgently needed. Beneficial effects of mesenchymal stem cells (MSC) have been described, however it is unclear whether the MSCs themselves or their secretome is required. We hypothesized that MSC-derived conditioned medium (CM) reduces progression of CKD and studied functional and structural effects in a rat model of established CKD. CKD was induced by 5/6 nephrectomy (SNX) combined with L-NNA and 6% NaCl diet in Lewis rats. Six weeks after SNX, CKD rats received either 50 µg CM or 50 µg non-CM (NCM) twice daily intravenously for four consecutive days. Six weeks after treatment CM administration was functionally effective: glomerular filtration rate (inulin clearance) and effective renal plasma flow (PAH clearance) were significantly higher in CM vs. NCM-treatment. Systolic blood pressure was lower in CM compared to NCM. Proteinuria tended to be lower after CM. Tubular and glomerular damage were reduced and more glomerular endothelial cells were found after CM. DNA damage repair was increased after CM. MSC-CM derived exosomes, tested in the same experimental setting, showed no protective effect on the kidney. In a rat model of established CKD, we demonstrated that administration of MSC-CM has a long-lasting therapeutic rescue function shown by decreased progression of CKD and reduced hypertension and glomerular injury.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038746PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378606PMC
December 2012

Cellular stress conditions are reflected in the protein and RNA content of endothelial cell-derived exosomes.

J Extracell Vesicles 2012 16;1. Epub 2012 Apr 16.

Department of Nephrology and Hypertension, UMC Utrecht, Utrecht, The Netherlands.

Background: The healthy vascular endothelium, which forms the barrier between blood and the surrounding tissues, is known to efficiently respond to stress signals like hypoxia and inflammation by adaptation of cellular physiology and the secretion of (soluble) growth factors and cytokines. Exosomes are potent mediators of intercellular communication. Their content consists of RNA and proteins from the cell of origin, and thus depends on the condition of these cells at the time of exosome biogenesis. It has been suggested that exosomes protect their target cells from cellular stress through the transfer of RNA and proteins. We hypothesized that endothelium-derived exosomes are involved in the endothelial response to cellular stress, and that exosome RNA and protein content reflect the effects of cellular stress induced by hypoxia, inflammation or hyperglycemia.

Methods: We exposed cultured endothelial cells to different types of cellular stress (hypoxia, TNF-α-induced activation, high glucose and mannose concentrations) and compared mRNA and protein content of exosomes produced by these cells by microarray analysis and a quantitative proteomics approach.

Results: We identified 1,354 proteins and 1,992 mRNAs in endothelial cell-derived exosomes. Several proteins and mRNAs showed altered abundances after exposure of their producing cells to cellular stress, which were confirmed by immunoblot or qPCR analysis.

Conclusion: Our data show that hypoxia and endothelial activation are reflected in RNA and protein exosome composition, and that exposure to high sugar concentrations alters exosome protein composition only to a minor extend, and does not affect exosome RNA composition.
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http://dx.doi.org/10.3402/jev.v1i0.18396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760650PMC
September 2013

Quantitative proteomic identification of host factors involved in the Salmonella typhimurium infection cycle.

Proteomics 2011 Dec 28;11(23):4477-91. Epub 2011 Oct 28.

Department of Biochemistry and Cell Biology, Biochemistry Division, Utrecht University, Utrecht, The Netherlands.

To identify host factors involved in Salmonella replication, SILAC-based quantitative proteomics was used to investigate the interactions of Salmonella typhimurium with the secretory pathway in human epithelial cells. Protein profiles of Golgi-enriched fractions isolated from S. typhimurium-infected cells were compared with those of mock-infected cells, revealing significant depletion or enrichment of 105 proteins. Proteins annotated to play a role in membrane traffic were overrepresented among the depleted proteins whereas proteins annotated to the cytoskeleton showed a diverse behavior with some proteins being enriched, others being depleted from the Golgi fraction upon Salmonella infection. To study the functional relevance of identified proteins in the Salmonella infection cycle, small interfering RNA (siRNA) experiments were performed. siRNA-mediated depletion of a selection of affected proteins identified five host factors involved in Salmonella infection. Depletion of peroxiredoxin-6 (PRDX6), isoform β-4c of integrin β-4 (ITGB4), isoform 1 of protein lap2 (erbin interacting protein; ERBB2IP), stomatin (STOM) or TBC domain containing protein 10b (TBC1D10B) resulted in increased Salmonella replication. Surprisingly, in addition to the effect on Salmonella replication, depletion of STOM or ITGB4 resulted in a dispersal of intracellular Salmonella microcolonies. It can be concluded that by using SILAC-based quantitative proteomics we were able to identify novel host cell proteins involved in the complex interplay between Salmonella and epithelial cells.
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http://dx.doi.org/10.1002/pmic.201100224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167899PMC
December 2011

Exosomes and the kidney: prospects for diagnosis and therapy of renal diseases.

Kidney Int 2011 Dec 31;80(11):1138-45. Epub 2011 Aug 31.

Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.

Exosomes are 40-100 nm membrane vesicles secreted into the extracellular space by numerous cell types. These structures can be isolated from body fluids including urine and plasma. Exosomes contain proteins, mRNAs, miRNAs, and signaling molecules that reflect the physiological state of their cells of origin and consequently provide a rich source of potential biomarker molecules. Aside from diagnostic uses, exosome-mediated transfer of proteins, mRNAs, miRNAs, and signaling molecules offer the promise that they may be used for therapeutic purposes. In this review, we integrate new knowledge about exosomes from outside the field of nephrology with recent progress by renal researchers in order to provide a basis for speculation about how the study of exosomes may affect the fields of nephrology and renal physiology in the next few years.
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http://dx.doi.org/10.1038/ki.2011.292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412193PMC
December 2011

The proteome of the insoluble Schistosoma mansoni eggshell skeleton.

Int J Parasitol 2011 Apr 12;41(5):523-32. Epub 2011 Jan 12.

Department of Medical Microbiology and Infectious Diseases, Erasmus MC, 's-Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands.

In schistosomiasis, the majority of symptoms of the disease is caused by the eggs that are trapped in the liver. These eggs elicit an immune reaction that leads to the formation of granulomas. The eggshell, which is a rigid insoluble structure built from cross-linked proteins, is the site of direct interaction between the egg and the immune system. However, the exact protein composition of the insoluble eggshell was previously unknown. To identify the proteins of the eggshell of Schistosoma mansoni we performed LC-MS/MS analysis, immunostaining and amino acid analysis on eggshell fragments. For this, eggshell protein skeleton was prepared by thoroughly cleaning eggshells in a four-step stripping procedure of increasing strength including urea and SDS to remove all material that is not covalently linked to the eggshell itself, but is part of the inside of the egg, such as Reynold's layer, von Lichtenberg's envelope and the miracidium. We identified 45 proteins of which the majority are non-structural proteins and non-specific for eggs, but are house-keeping proteins that are present in large quantities in worms and miracidia. Some of these proteins are known to be immunogenic, such as HSP70, GST and enolase. In addition, a number of schistosome-specific proteins with unknown function and no homology to any known annotated protein were found to be incorporated in the eggshell. Schistosome-specific glycoconjugates were also shown to be present on the eggshell protein skeleton. This study also confirmed that the putative eggshell protein p14 contributes largely to the eggshell. Together, these results give new insights into eggshell composition as well as eggshell formation. Those proteins that are present at the site and time of eggshell formation are incorporated in the cross-linked eggshell and this cross-linking does no longer occur when the miracidium starts secreting proteins.
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http://dx.doi.org/10.1016/j.ijpara.2010.12.005DOI Listing
April 2011

Identification of host factors involved in coronavirus replication by quantitative proteomics analysis.

Proteomics 2011 Jan 6;11(1):64-80. Epub 2010 Dec 6.

Department of Biochemistry and Cell Biology, Biochemistry Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

In this study, we applied a quantitative proteomic approach, based on SILAC, to investigate the interactions of coronaviruses with the secretory pathway of the host cell, with the aim to identify host factors involved in coronavirus replication. Comparison of the protein profiles of Golgi-enriched fractions of cells that were either mock infected or infected with mouse hepatitis virus revealed the significant depletion or enrichment of 116 proteins. Although ribosomal/nucleic acid binding proteins were enriched in the Golgi-fractions of mouse hepatitis virus-infected cells, proteins annotated to localize to several organelles of the secretory pathway were overrepresented among the proteins that were depleted from these fractions upon infection. We hypothesized that proteins, of which the abundance or distribution is affected by infection, are likely to be involved in the virus life cycle. Indeed, depletion of a small subset of the affected proteins by using small interfering RNAs identified several host factors involved in coronavirus infection. Transfection of small interfering RNAs targeting either C11orf59 or Golgi apparatus glycoprotein 1 resulted in increased virus replication, whereas depletion of vesicle-trafficking protein vesicle-trafficking protein sec22b enhanced the release of infectious progeny virus. Overexpression of these proteins, on the other hand, had a negative effect on virus replication. Overall, our study shows that the SILAC approach is a suitable tool to study host-pathogen interactions and to identify host proteins involved in virus replication.
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http://dx.doi.org/10.1002/pmic.201000309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167679PMC
January 2011

Transcriptome analysis in endothelial progenitor cell biology.

Antioxid Redox Signal 2011 Aug 14;15(4):1029-42. Epub 2011 Feb 14.

Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.

The use of endothelial progenitor cells (EPCs) is a promising new treatment option for cardiovascular diseases. Many of the underlying mechanisms that result in an improvement of endothelial function in vivo remain poorly elucidated to this date, however. We summarize the current positions and potential applications of gene-expression profiling in the field of EPC biology. Based on our own and published gene-expression data, we demonstrate that gene-expression profiling can efficiently be used to characterize different EPC types. Furthermore, we highlight the potential of gene-expression profiling for the analysis of changes that EPCs undergo during culture and examine changes in gene transcription in diseased patients. Transcriptome profiling is a powerful tool for the characterization and functional analysis of EPCs in health and disease.
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http://dx.doi.org/10.1089/ars.2010.3594DOI Listing
August 2011

MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis.

Immunol Cell Biol 2010 Nov-Dec;88(8):851-6. Epub 2010 May 11.

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands.

Professional antigen-presenting cells secrete major histocompatibility complex class II (MHC II) carrying exosomes with unclear physiological function(s). Exosomes are first generated as the intraluminal vesicles (ILVs) of a specific type of multivesicular body, and are then secreted by fusion of this compartment with the plasma membrane. We have previously shown that in contrast to the sorting of MHC II at lysosomally targeted multivesicular bodies, sorting of MHC II into exosomes does not rely on MHC II ubiquitination. In search for proteins that drive the incorporation of MHC II into exosomes or functionally discriminate exosomal from plasma membrane MHC II, we first analyzed the total proteome of highly purified B cell-derived exosomes using sensitive and accurate mass spectrometry (MS), and identified 539 proteins, including known and not previously identified constituents. Using quantitative MS, we then identified a small subset of proteins that were specifically co-immunoprecipitated with MHC II from detergent-solubilized exosomes. These include HSC71, HSP90, 14-3-3ɛ, CD20 and pyruvate kinase type M2 (PKM2), and we speculate on the functionality of their interaction with exosomal MHC II.
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http://dx.doi.org/10.1038/icb.2010.64DOI Listing
June 2011