Publications by authors named "Jiong-Wei Wang"

35 Publications

Therapeutic and diagnostic targeting of fibrosis in metabolic, proliferative and viral disorders.

Adv Drug Deliv Rev 2021 Jun 14:113831. Epub 2021 Jun 14.

Department of Nanomedicine and Theranostics, Institute for Experimental Molecular Imaging, Faculty of Medicine, RWTH Aachen University, Aachen, Germany; Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands; Department of Targeted Therapeutics, University of Twente, Enschede, The Netherlands. Electronic address:

Fibrosis is a common denominator in many pathologies and crucially affects disease progression, drug delivery efficiency and therapy outcome. We here summarize therapeutic and diagnostic strategies for fibrosis targeting in atherosclerosis and cardiac disease, cancer, diabetes, liver diseases and viral infections. We address various anti-fibrotic targets, ranging from cells and genes to metabolites and proteins, primarily focusing on fibrosis-promoting features that are conserved among the different diseases. We discuss how anti-fibrotic therapies have progressed over the years, and how nanomedicine formulations can potentiate anti-fibrotic treatment efficacy. From a diagnostic point of view, we discuss how medical imaging can be employed to facilitate the diagnosis, staging and treatment monitoring of fibrotic disorders. Altogether, this comprehensive overview serves as a basis for developing individualized and improved treatment strategies for patients suffering from fibrosis-associated pathologies.
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http://dx.doi.org/10.1016/j.addr.2021.113831DOI Listing
June 2021

Extracellular vesicles as a drug delivery system: A systematic review of preclinical studies.

Adv Drug Deliv Rev 2021 May 18;175:113801. Epub 2021 May 18.

Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 119228 Singapore, Singapore; Cardiovascular Research Institute (CVRI), National University Heart Centre Singapore (NUHCS), 117599 Singapore, Singapore; Nanomedicine Translational Research Programme, Centre for NanoMedicine, Yong Loo Lin School of Medicine, National University of Singapore, 117609 Singapore, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore. Electronic address:

During the past decades, extracellular vesicles (EVs) have emerged as an attractive drug delivery system. Here, we assess their pre-clinical applications, in the form of a systematic review. For each study published in the past decade, disease models, animal species, EV donor cell types, active pharmaceutical ingredients (APIs), EV surface modifications, API loading methods, EV size and charge, estimation of EV purity, presence of biodistribution studies and administration routes were quantitatively analyzed in a defined and reproducible way. We have interpreted the trends we observe over the past decade, to define the niches where to apply EVs for drug delivery in the future and to provide a basis for regulatory guidelines.
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http://dx.doi.org/10.1016/j.addr.2021.05.011DOI Listing
May 2021

Enhancing the cardiovascular protective effects of a healthy dietary pattern with wolfberry (Lycium barbarum): A randomized controlled trial.

Am J Clin Nutr 2021 May 8. Epub 2021 May 8.

Department of Food Science & Technology, Faculty of Science, National University of Singapore, Singapore, Singapore.

Background: The consumption of wolfberry (Lycium barbarum), a rich source of carotenoids and bioactive polysaccharides, may serve as a potential dietary strategy for cardiovascular disease (CVD) risk management although limited studies examined its effects as whole fruits.

Objectives: To investigate the impact of wolfberry consumption as part of a healthy dietary pattern on vascular health-related outcomes and classical CVD risk factors in middle-aged and older adults in Singapore.

Methods: This is a 16-week, parallel design, randomized controlled trial. All participants (n = 40) received dietary counselling to follow healthy dietary pattern recommendations with the wolfberry group given additional instructions to cook and consume 15 g/d whole, dried wolfberry with their main meals. Biomarkers of vascular function (flow-mediated dilation, plasma total nitrate/nitrite, endothelin-1, and intercellular adhesion molecule-1), vascular structure (carotid intima-media thickness) and vascular regeneration (endothelial progenitor cell count, plasma angiopoietin 1 and angiopoietin 2), were assessed at baseline and postintervention. Serum lipid-lipoproteins and blood pressure were evaluated every 4 weeks.

Results: All participants showed an improved compliance toward the healthy dietary pattern. This was coupled with marked rises in total nitrate/nitrite concentrations (mean change wolfberry: 3.92 ± 1.73 nmol/mL; control: 5.01 ± 2.55 nmol/L) and reductions in endothelin-1 concentrations (wolfberry: -0.19 ± 0.06 pg/mL; control: -0.15 ± 0.08 pg/mL). Compared with the control which depicted no changes from baseline, the wolfberry group had a significantly higher HDL cholesterol (0.08 ± 0.04 mmol/L), as well as lower Framingham predicted long-term CVD risk (-0.8 ± 0.5%) and vascular age (-1.9 ± 1.0 y) postintervention. No differences were observed in the other vascular health-related outcomes.

Conclusions: In middle-aged and older adults, adherence to a healthy dietary pattern improves vascular tone. Incorporating wolfberry to the diet further improves blood lipid-lipoprotein profile and may lower long-term CVD risk. This study was registered at clinicatrials.gov as NCT03535844.
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http://dx.doi.org/10.1093/ajcn/nqab062DOI Listing
May 2021

Capturing Cytokines with Advanced Materials: A Potential Strategy to Tackle COVID-19 Cytokine Storm.

Adv Mater 2021 May 10;33(20):e2100012. Epub 2021 Apr 10.

Institute of Biomedical Health Technology and Engineering, Shenzhen Bay Laboratory, Shenzhen, 518132, China.

The COVID-19 pandemic, induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused great impact on the global economy and people's daily life. In the clinic, most patients with COVID-19 show none or mild symptoms, while approximately 20% of them develop severe pneumonia, multiple organ failure, or septic shock due to infection-induced cytokine release syndrome (the so-called "cytokine storm"). Neutralizing antibodies targeting inflammatory cytokines may potentially curb immunopathology caused by COVID-19; however, the complexity of cytokine interactions and the multiplicity of cytokine targets make attenuating the cytokine storm challenging. Nonspecific in vivo biodistribution and dose-limiting side effects further limit the broad application of those free antibodies. Recent advances in biomaterials and nanotechnology have offered many promising opportunities for infectious and inflammatory diseases. Here, potential mechanisms of COVID-19 cytokine storm are first discussed, and relevant therapeutic strategies and ongoing clinical trials are then reviewed. Furthermore, recent research involving emerging biomaterials for improving antibody-based and broad-spectrum cytokine neutralization is summarized. It is anticipated that this work will provide insights on the development of novel therapeutics toward efficacious management of COVID-19 cytokine storm and other inflammatory diseases.
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http://dx.doi.org/10.1002/adma.202100012DOI Listing
May 2021

Extracellular Vesicle (EV) biohybrid systems for cancer therapy: Recent advances and future perspectives.

Semin Cancer Biol 2021 Feb 17. Epub 2021 Feb 17.

Department of Pharmacy, National University of Singapore, Singapore. Electronic address:

Extracellular vesicles (EVs) are a class of cell-derived lipid-bilayer membrane vesicles secreted by almost all mammalian cells and involved in intercellular communication by shuttling various biological cargoes. Over the last decade, EVs - namely exosomes and microvesicles - have been extensively explored as next-generation nanoscale drug delivery systems (DDSs). This is in large due to their endogenous origin, which enables EVs to circumvent some of the limitations associated with existing cancer therapy approaches (i.e. by preventing recognition by the immune system and improving selectivity towards tumor tissue). However, successful translation of these cell-derived vesicles into clinical applications has been hindered by several factors, among which the loading of exogenous therapeutic molecules still represents a great challenge. In order to address this issue and to further advance these biologically-derived systems as drug carriers, EV-biohybrid nano-DDSs, obtained through the fusion of EVs with conventional synthetic nano-DDSs, have recently been proposed as a valuable alternative as DDSs. Building on the idea of "combining the best of both worlds", a combination of these two unique entities aims to harness the beneficial properties associated with both EVs and conventional nano-DDSs, while overcoming the flaws of the individual components. These biohybrid systems also provide a unique opportunity for exploitation of new synergisms, often leading to improved therapeutic outcomes, thus paving the way for advancements in cancer therapy. This review aims to describe the recent developments of EV-biohybrid nano-DDSs in cancer therapy, to highlight the most promising results and breakthroughs, as well as to provide a glimpse on the possible intrinsic targeting mechanisms of EVs that can be bequeathed to their hybrid systems. Finally, we also provide some insights in the future perspectives of EV-hybrid DDSs.
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http://dx.doi.org/10.1016/j.semcancer.2021.02.006DOI Listing
February 2021

In Vivo Three-Photon Imaging of Lipids using Ultrabright Fluorogens with Aggregation-Induced Emission.

Adv Mater 2021 Mar 11;33(11):e2007490. Epub 2021 Feb 11.

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore.

Fluorescent probes capable of in vivo lipids labeling are highly desirable for studying lipid-accumulation-related metabolic diseases, such as nonalcoholic fatty liver disease, type-2 diabetes, and atherosclerosis. However, most of the current lipid-specific fluorophores cannot be used for in vivo labeling due to their strong hydrophobicity. Herein, organic dots from bright luminogens with aggregation-induced emission (AIEgen) are developed for in vivo labeling and three-photon fluorescence imaging of lipid-rich tissues, such as fatty liver, atherosclerotic plaques in brain vasculatures, and carotid arteries. The organic dots show excellent stability in an aqueous medium with high targeting specificity to lipids and strong three-photon fluorescence in the far-red/near-infrared (NIR) region under NIR-II laser excitation, which enables efficient in vivo labeling and imaging of lipids in deep tissues. The study will inspire the development of lipid-targeting fluorophores for in vivo applications.
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http://dx.doi.org/10.1002/adma.202007490DOI Listing
March 2021

Hypoxia-induced amniotic fluid stem cell secretome augments cardiomyocyte proliferation and enhances cardioprotective effects under hypoxic-ischemic conditions.

Sci Rep 2021 01 8;11(1):163. Epub 2021 Jan 8.

Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Secretome derived from human amniotic fluid stem cells (AFSC-S) is rich in soluble bioactive factors (SBF) and offers untapped therapeutic potential for regenerative medicine while avoiding putative cell-related complications. Characterization and optimal generation of AFSC-S remains challenging. We hypothesized that modulation of oxygen conditions during AFSC-S generation enriches SBF and confers enhanced regenerative and cardioprotective effects on cardiovascular cells. We collected secretome at 6-hourly intervals up to 30 h following incubation of AFSC in normoxic (21%O, nAFSC-S) and hypoxic (1%O, hAFSC-S) conditions. Proliferation of human adult cardiomyocytes (hCM) and umbilical cord endothelial cells (HUVEC) incubated with nAFSC-S or hAFSC-S were examined following culture in normoxia or hypoxia. Lower AFSC counts and richer protein content in AFSC-S were observed in hypoxia. Characterization of AFSC-S by multiplex immunoassay showed higher concentrations of pro-angiogenic and anti-inflammatory SBF. hCM demonstrated highest proliferation with 30h-hAFSC-S in hypoxic culture. The cardioprotective potential of concentrated 30h-hAFSC-S treatment was demonstrated in a myocardial ischemia-reperfusion injury mouse model by infarct size and cell apoptosis reduction and cell proliferation increase when compared to saline treatment controls. Thus, we project that hypoxic-generated AFSC-S, with higher pro-angiogenic and anti-inflammatory SBF, can be harnessed and refined for tailored regenerative applications in ischemic cardiovascular disease.
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http://dx.doi.org/10.1038/s41598-020-80326-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794288PMC
January 2021

Cell-Derived Nanovesicles as Exosome-Mimetics for Drug Delivery Purposes: Uses and Recommendations.

Methods Mol Biol 2021 ;2211:147-170

Department of Pharmacy, National University of Singapore, Singapore, Singapore.

Cell-derived Drug Delivery Systems (DDSs), particularly exosomes, have grown in popularity and have been increasingly explored as novel DDSs, due to their intrinsic targeting capabilities. However, clinical translation of exosomes is impeded by the tedious isolation procedures and poor yield. Cell-derived nanovesicles (CDNs) have recently been produced and proposed as exosome-mimetics. Various methods for producing exosome-mimetics have been developed. In this chapter, we present a simple, efficient, and cost-effective CDNs production method that uses common laboratory equipment (microcentrifuge) and spin cups. Through a series of extrusion and size exclusion steps, CDNs are produced from in vitro cell culture and are found to highly resemble the endogenous exosomes. Thus, we envision that this strategy holds great potential as a viable alternative to exosomes in the development of ideal DDS.
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http://dx.doi.org/10.1007/978-1-0716-0943-9_11DOI Listing
March 2021

Micro cell vesicle technology (mCVT): a novel hybrid system of gene delivery for hard-to-transfect (HTT) cells.

Nanoscale 2020 Sep 28;12(35):18022-18030. Epub 2020 Aug 28.

Department of Pharmacy, National University of Singapore, Singapore.

A hybrid gene delivery platform, micro Cell Vesicle Technology (mCVT), produced from the fusion of plasma membranes and cationic lipids, is presently used to improve the transfection efficiency of hard-to-transfect (HTT) cells. The plasma membrane components of mCVTs impart specificity in cellular uptake and reduce cytotoxicity in the transfection process, while the cationic lipids complex with the genetic material and provide structural integrity to mCVTs.
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http://dx.doi.org/10.1039/d0nr03784bDOI Listing
September 2020

Extracellular vesicle Cystatin C and CD14 are associated with both renal dysfunction and heart failure.

ESC Heart Fail 2020 10 10;7(5):2240-2249. Epub 2020 Jul 10.

Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

Aims: Extracellular vesicles (EVs) are small double-membrane plasma vesicles that play key roles in cellular crosstalk and mechanisms such as inflammation. The role of EVs in combined organ failure such as cardiorenal syndrome has not been investigated. The aim of this study is to identify EV proteins that are associated with renal dysfunction, heart failure, and their combination in dyspnoeic patients.

Methods And Results: Blood samples were prospectively collected in 404 patients presenting with breathlessness at the emergency department at National University Hospital, Singapore. Renal dysfunction was defined as estimated glomerular filtration rate < 60 mL/min/1.73 m . The presence of heart failure was independently adjudicated by two clinicians on the basis of the criteria of the European Society of Cardiology guidelines. Protein levels of SerpinG1, SerpinF2, Cystatin C, and CD14 were measured with a quantitative immune assay within three EV sub-fractions and in plasma and were tested for their associations with renal dysfunction, heart failure, and the concurrence of both conditions using multinomial regression analysis, thereby correcting for confounders such as age, gender, ethnicity, and co-morbidities. Renal dysfunction was found in 92 patients (23%), while heart failure was present in 141 (35%). In total, 58 patients (14%) were diagnosed with both renal dysfunction and heart failure. Regression analysis showed that Cystatin C was associated with renal dysfunction, heart failure, and their combination in all three EV sub-fractions and in plasma. CD14 was associated with both renal dysfunction and the combined renal dysfunction and heart failure in all EV sub-fractions, and with presence of heart failure in the high density lipoprotein sub-fraction. SerpinG1 and SerpinF2 were associated with heart failure in, respectively, two and one out of three EV sub-fractions and in plasma, but not with renal dysfunction.

Conclusions: We provide the first data showing that Cystatin C and CD14 in circulating EVs are associated with both renal dysfunction and heart failure in patients presenting with acute dyspnoea. This suggests that EV proteins may be involved in the combined organ failure of the cardiorenal syndrome and may represent possible targets for prevention or treatment.
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http://dx.doi.org/10.1002/ehf2.12699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524227PMC
October 2020

Magnetic fields modulate metabolism and gut microbiome in correlation with Pgc-1α expression: Follow-up to an in vitro magnetic mitohormetic study.

FASEB J 2020 08 6;34(8):11143-11167. Epub 2020 Jul 6.

Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Exercise modulates metabolism and the gut microbiome. Brief exposure to low mT-range pulsing electromagnetic fields (PEMFs) was previously shown to accentuate in vitro myogenesis and mitochondriogenesis by activating a calcium-mitochondrial axis upstream of PGC-1α transcriptional upregulation, recapitulating a genetic response implicated in exercise-induced metabolic adaptations. We compared the effects of analogous PEMF exposure (1.5 mT, 10 min/week), with and without exercise, on systemic metabolism and gut microbiome in four groups of mice: (a) no intervention; (b) PEMF treatment; (c) exercise; (d) exercise and PEMF treatment. The combination of PEMFs and exercise for 6 weeks enhanced running performance and upregulated muscular and adipose Pgc-1α transcript levels, whereas exercise alone was incapable of elevating Pgc-1α levels. The gut microbiome Firmicutes/Bacteroidetes ratio decreased with exercise and PEMF exposure, alone or in combination, which has been associated in published studies with an increase in lean body mass. After 2 months, brief PEMF treatment alone increased Pgc-1α and mitohormetic gene expression and after >4 months PEMF treatment alone enhanced oxidative muscle expression, fatty acid oxidation, and reduced insulin levels. Hence, short-term PEMF treatment was sufficient to instigate PGC-1α-associated transcriptional cascades governing systemic mitohormetic adaptations, whereas longer-term PEMF treatment was capable of inducing related metabolic adaptations independently of exercise.
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http://dx.doi.org/10.1096/fj.201903005RRDOI Listing
August 2020

In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors.

Cell Rep 2020 May;31(8):107714

Cardiovascular & Metabolic Disorders Program, Duke-NUS Medical School, National University of Singapore, Singapore 169857, Singapore; Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden. Electronic address:

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http://dx.doi.org/10.1016/j.celrep.2020.107714DOI Listing
May 2020

Predicting human pharmacokinetics of liposomal temoporfin using a hybrid in silico model.

Eur J Pharm Biopharm 2020 Apr 6;149:121-134. Epub 2020 Feb 6.

Department of Pharmacy, Faculty of Science, National University of Singapore, 6 Science Drive 2, Singapore 117546, Singapore. Electronic address:

Over the years, the performance of the liposomal formulations of temoporfin, Foslip® and Fospeg®, was investigated in a broad array of cell-based assays and preclinical animal models. So far, little attention has been paid to the influence of drug release and liposomal stability on the plasma concentration-time profile. The drug release is a key attribute which impacts product quality and the in vivo efficacy of nanocarrier formulations. In the present approach, the in vitro drug release and the drug-protein transfer of Foslip® and Fospeg® was determined using the dispersion releaser technology. To analyze the stability of both formulations in physiological fluids, nanoparticle tracking analysis was applied. A comparable drug release behavior and a high physical stability with a vesicle size of approximately 92 ± 2 nm for Foslip® and at 111 ± 5 nm for Fospeg® were measured. The development of a novel hybrid in silico model resulted in an optimal representation of the in vivo data. Based on the information available for previous formulations, the model enabled a prediction of the performance of Foslip® in humans. To verify the simulations, plasma concentration-time profiles of a phase I clinical trial were used. An absolute average fold error of 1.4 was achieved. Moreover, a deconvolution of the pharmacokinetic profile into different fractions relevant for the in vivo efficacy and safety was achieved. While the total plasma concentration reached a c of 2298 ng/mL after 0.72 h, the monomolecular drug accounted for a small fraction of the photosensitizer with a c of 321 ng/mL only.
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http://dx.doi.org/10.1016/j.ejpb.2020.02.001DOI Listing
April 2020

Yin Yang 1 Suppresses Dilated Cardiomyopathy and Cardiac Fibrosis Through Regulation of and .

Circ Res 2019 10 9;125(9):834-846. Epub 2019 Sep 9.

From the Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (C.Y.T., J.X.W., P.S.C., H.T., D.L., W.C., J.J.).

Rationale: Pathogenic variations in the lamin gene () cause familial dilated cardiomyopathy (DCM). insufficiency caused by pathogenic variants is believed to be the basic mechanism underpinning -related DCM.

Objective: To assess whether silencing of cardiac causes DCM and investigate the role of Yin Yang 1 () in suppressing DCM.

Methods And Results: We developed a DCM mouse model induced by cardiac-specific short hairpin RNA. Silencing of cardiac induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of suppressed DCM and cardiac fibrosis by inducing expression and preventing upregulation of . Knockdown of upregulated attenuated the suppressive effect of on DCM and cardiac fibrosis. However, upregulation of alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of together with silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of regulated and reporter activities and modulated and gene expression in cardiomyocytes. Downregulation of inhibited TGF-β (transforming growth factor-β)/Smad signaling in DCM hearts. Regulation of both and further suppressed TGFβ/Smad signaling. In addition, co-modulation of and reduced CD3+ T cell numbers in DCM hearts.

Conclusions: Our findings demonstrate that upregulation of or co-modulation of and offer novel therapeutic strategies for the treatment of DCM caused by insufficiency.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.314794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336364PMC
October 2019

Extracellular Vesicles in Cardiovascular Diseases: Alternative Biomarker Sources, Therapeutic Agents, and Drug Delivery Carriers.

Int J Mol Sci 2019 Jul 3;20(13). Epub 2019 Jul 3.

Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore.

Cardiovascular diseases (CVD) represent the leading cause of morbidity and mortality globally. The emerging role of extracellular vesicles (EVs) in intercellular communication has stimulated renewed interest in exploring the potential application of EVs as tools for diagnosis, prognosis, and therapy in CVD. The ubiquitous nature of EVs in biological fluids presents a technological advantage compared to current diagnostic tools by virtue of their notable stability. EV contents, such as proteins and microRNAs, represent specific signatures of cellular activation or injury. This feature positions EVs as an alternative source of biomarkers. Furthermore, their intrinsic activity and immunomodulatory properties offer EVs unique opportunities to act as therapeutic agents per se or to serve as drug delivery carriers by acting as miniaturized vehicles incorporating bioactive molecules. In this article, we aim to review the recent advances and applications of EV-based biomarkers and therapeutics. In addition, the potential of EVs as a drug delivery and theranostic platform for CVD will also be discussed.
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http://dx.doi.org/10.3390/ijms20133272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650854PMC
July 2019

In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors.

Cell Rep 2019 03;26(12):3231-3245.e9

Cardiovascular & Metabolic Disorders Program, Duke-NUS Medical School, National University of Singapore, Singapore 169857, Singapore; Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden. Electronic address:

Regeneration of injured human heart muscle is limited and an unmet clinical need. There are no methods for the reproducible generation of clinical-quality stem cell-derived cardiovascular progenitors (CVPs). We identified laminin-221 (LN-221) as the most likely expressed cardiac laminin. We produced it as human recombinant protein and showed that LN-221 promotes differentiation of pluripotent human embryonic stem cells (hESCs) toward cardiomyocyte lineage and downregulates pluripotency and teratoma-associated genes. We developed a chemically defined, xeno-free laminin-based differentiation protocol to generate CVPs. We show high reproducibility of the differentiation protocol using time-course bulk RNA sequencing developed from different hESC lines. Single-cell RNA sequencing of CVPs derived from hESC lines supported reproducibility and identified three main progenitor subpopulations. These CVPs were transplanted into myocardial infarction mice, where heart function was measured by echocardiogram and human heart muscle bundle formation was identified histologically. This method may provide clinical-quality cells for use in regenerative cardiology.
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http://dx.doi.org/10.1016/j.celrep.2019.02.083DOI Listing
March 2019

Toll-like receptor 7 deficiency promotes survival and reduces adverse left ventricular remodelling after myocardial infarction.

Cardiovasc Res 2019 Oct;115(12):1791-1803

Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Aims: The Toll-like receptor 7 (TLR7) is an intracellular innate immune receptor activated by nucleic acids shed from dying cells leading to activation of the innate immune system. Since innate immune system activation is involved in the response to myocardial infarction (MI), this study aims to identify if TLR7 is involved in post-MI ischaemic injury and adverse remodelling after MI.

Methods And Results: TLR7 involvement in MI was investigated in human tissue from patients with ischaemic heart failure, as well as in a mouse model of permanent left anterior descending artery occlusion in C57BL/6J wild type and TLR7 deficient (TLR7-/-) mice. TLR7 expression was up-regulated in human and mouse ischaemic myocardium after MI. Compared to wild type mice, TLR7-/- mice had less acute cardiac rupture associated with blunted activation of matrix metalloproteinase 2, increased expression of tissue inhibitor of metalloproteinase 1, recruitment of more myofibroblasts, and the formation of a myocardial scar with higher collagen fibre density. Furthermore, inflammatory cell influx and inflammatory cytokine expression post-MI were reduced in the TLR7-/- heart. During a 28-day follow-up after MI, TLR7 deficiency resulted in less chronic adverse left ventricular remodelling and better cardiac function. Bone marrow (BM) transplantation experiments showed that TLR7 deficiency in BM-derived cells preserved cardiac function after MI.

Conclusions: In acute MI, TLR7 mediates the response to acute cardiac injury and chronic remodelling probably via modulation of post-MI scar formation and BM-derived inflammatory infiltration of the myocardium.
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http://dx.doi.org/10.1093/cvr/cvz057DOI Listing
October 2019

LDL extracellular vesicle coagulation protein levels change after initiation of statin therapy. Findings from the METEOR trial.

Int J Cardiol 2018 Nov 26;271:247-253. Epub 2018 May 26.

Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore & Cardiovascular Research Institute, National University Heart Centre Singapore, Singapore; Experimental Cardiology Laboratory, University Medical Center Utrecht, Utrecht University, The Netherlands; Interuniversity Cardiology Institute of the Netherlands, Utrecht University, The Netherlands; Department of Vascular Surgery, University Medical Center Utrecht, Utrecht University, The Netherlands. Electronic address:

Background: Statins are thought to have pleiotropic properties, including anticoagulant effects, in addition to reducing lipoprotein (LDL) levels. Plasma extracellular vesicles (EVs) are small bilayer membrane vesicles involved in various biological processes including coagulation. Since subsets of EVs in the LDL plasma fraction (LDL-EVs) correlate with thrombin activity, we hypothesized that changes in LDL-EVs after statin therapy may differ from that of serum levels of coagulation proteins, providing insight into the effects of statins on coagulation.

Methods: The study was conducted in 666 subjects with available serum from the METEOR trial, a trial of the effect of rosuvastatin versus placebo in patients with subclinical atherosclerosis. Changes in protein levels of von Willebrand Factor (VWF), SerpinC1 and plasminogen were measured in serum and in LDL-EVs, and were compared between the rosuvastatin and placebo groups.

Results: LDL-EV levels of plasminogen and VWF increased with rosuvastatin treatment compared to placebo (mean change of 126 ± 8 versus 17 ± 12 μg/mL for plasminogen (p < 0.001) and 310 ± 60 versus 64 ± 55 μg/mL for VWF (p = 0.015)). There was no difference between groups for change in LDL-EV-SerpinC1. In contrast, serum plasminogen levels increased to a lesser extent with rosuvastatin compared to placebo (23 ± 29 versus 67 ± 17 μg/mL, p = 0.024) and serum VWF levels showed no significant difference between both groups.

Conclusions: Rosuvastatin increases LDL-EV coagulation proteins plasminogen and VWF in patients with subclinical atherosclerosis, an effect that is different from the effect of rosuvastatin on the same proteins in serum. This identifies LDL-EVs as a newly detected possible intermediate between statin therapy and coagulation.
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http://dx.doi.org/10.1016/j.ijcard.2018.05.098DOI Listing
November 2018

Regulation of Blood Pressure by Targeting Ca1.2-Galectin-1 Protein Interaction.

Circulation 2018 10;138(14):1431-1445

Department of Physiology, Yong Loo Lin School of Medicine (Z.Y.H., J.-W.W., D.Y., M.C.L., Y.P.W., T.W.S.), National University of Singapore.

Background: L-type Ca1.2 channels play crucial roles in the regulation of blood pressure. Galectin-1 (Gal-1) has been reported to bind to the I-II loop of Ca1.2 channels to reduce their current density. However, the mechanistic understanding for the downregulation of Ca1.2 channels by Gal-1 and whether Gal-1 plays a direct role in blood pressure regulation remain unclear.

Methods: In vitro experiments involving coimmunoprecipitation, Western blot, patch-clamp recordings, immunohistochemistry, and pressure myography were used to evaluate the molecular mechanisms by which Gal-1 downregulates Ca1.2 channel in transfected, human embryonic kidney 293 cells, smooth muscle cells, arteries from Lgasl1 mice, rat, and human patients. In vivo experiments involving the delivery of Tat-e9c peptide and AAV5-Gal-1 into rats were performed to investigate the effect of targeting Ca1.2-Gal-1 interaction on blood pressure monitored by tail-cuff or telemetry methods.

Results: Our study reveals that Gal-1 is a key regulator for proteasomal degradation of Ca1.2 channels. Gal-1 competed allosterically with the Caβ subunit for binding to the I-II loop of the Ca1.2 channel. This competitive disruption of Caβ binding led to Ca1.2 degradation by exposing the channels to polyubiquitination. It is notable that we demonstrated that the inverse relationship of reduced Gal-1 and increased Ca1.2 protein levels in arteries was associated with hypertension in hypertensive rats and patients, and Gal-1 deficiency induces higher blood pressure in mice because of the upregulated Ca1.2 protein level in arteries. To directly regulate blood pressure by targeting the Ca1.2-Gal-1 interaction, we administered Tat-e9c, a peptide that competed for binding of Gal-1 by a miniosmotic pump, and this specific disruption of Ca1.2-Gal-1 coupling increased smooth muscle Ca1.2 currents, induced larger arterial contraction, and caused hypertension in rats. In contrasting experiments, overexpression of Gal-1 in smooth muscle by a single bolus of AAV5-Gal-1 significantly reduced blood pressure in spontaneously hypertensive rats.

Conclusions: We have defined molecularly that Gal-1 promotes Ca1.2 degradation by replacing Caβ and thereby exposing specific lysines for polyubiquitination and by masking I-II loop endoplasmic reticulum export signals. This mechanistic understanding provided the basis for targeting Ca1.2-Gal-1 interaction to demonstrate clearly the modulatory role that Gal-1 plays in regulating blood pressure, and offering a potential approach for therapeutic management of hypertension.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.031231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6185826PMC
October 2018

Lowering Low-Density Lipoprotein Particles in Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles.

Int J Mol Sci 2017 Dec 29;19(1). Epub 2017 Dec 29.

Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 119228 Singapore, Singapore.

Plasma extracellular vesicles (EVs) are lipid membrane vesicles involved in several biological processes including coagulation. Both coagulation and lipid metabolism are strongly associated with cardiovascular events. Lowering very-low- and low-density lipoprotein ((V)LDL) particles via dextran sulphate LDL apheresis also removes coagulation proteins. It remains unknown, however, how coagulation proteins are removed in apheresis. We hypothesize that plasma EVs that contain high levels of coagulation proteins are concomitantly removed with (V)LDL particles by dextran sulphate apheresis. For this, we precipitated (V)LDL particles from human plasma with dextran sulphate and analyzed the abundance of coagulation proteins and EVs in the precipitate. Coagulation pathway proteins, as demonstrated by proteomics and a bead-based immunoassay, were over-represented in the (V)LDL precipitate. In this precipitate, both bilayer EVs and monolayer (V)LDL particles were observed by electron microscopy. Separation of EVs from (V)LDL particles using density gradient centrifugation revealed that almost all coagulation proteins were present in the EVs and not in the (V)LDL particles. These EVs also showed a strong procoagulant activity. Our study suggests that dextran sulphate used in LDL apheresis may remove procoagulant EVs concomitantly with (V)LDL particles, leading to a loss of coagulation proteins from the blood.
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http://dx.doi.org/10.3390/ijms19010094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796044PMC
December 2017

EXOPLEXs: Chimeric Drug Delivery Platform from the Fusion of Cell-Derived Nanovesicles and Liposomes.

Biomacromolecules 2018 01 8;19(1):22-30. Epub 2017 Dec 8.

NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences (CeLS) , 28 Medical Drive, #05-01, Singapore 117456.

Cell-derived nanovesicles (CDNs) have been recently investigated as novel drug delivery systems (DDSs), due to the preservation of key features from the cell membrane of their precursor cells, which are responsible for an efficient cellular uptake by target cells. However, CDNs suffer from low drug loading efficiencies as well as challenges in functionalization compared to conventional DDS like liposomes. Here, we describe the first study proposing the fusion of CDNs with liposomes to form EXOPLEXs. We report the preservation of cell membranes from precursor cells similarly to CDNs, as well as high loading efficiencies of more than 65% with doxorubicin hydrochloride, a model chemotherapeutic drug. The doxorubicin-loaded EXOPLEXs (DOX-EXO) also demonstrated a higher in vitro cell killing effect than liposomes, while EXOPLEXs alone did not show any remarkable cytotoxicity. Taken together, these results illustrate the potential of EXOPLEXs as a novel DDS for targeted delivery of chemotherapeutics.
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http://dx.doi.org/10.1021/acs.biomac.7b01176DOI Listing
January 2018

Bioinspired Cell-Derived Nanovesicles versus Exosomes as Drug Delivery Systems: a Cost-Effective Alternative.

Sci Rep 2017 10 30;7(1):14322. Epub 2017 Oct 30.

NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences (CeLS), Singapore, Singapore.

Cell Derived Nanovesicles (CDNs) have been developed from the rapidly expanding field of exosomes, representing a class of bioinspired Drug Delivery Systems (DDS). However, translation to clinical applications is limited by the low yield and multi-step approach in isolating naturally secreted exosomes. Here, we show the first demonstration of a simple and rapid production method of CDNs using spin cups via a cell shearing approach, which offers clear advantages in terms of yield and cost-effectiveness over both traditional exosomes isolation, and also existing CDNs fabrication techniques. The CDNs obtained were of a higher protein yield and showed similarities in terms of physical characterization, protein and lipid analysis to both exosomes and CDNs previously reported in the literature. In addition, we investigated the mechanisms of cellular uptake of CDNs in vitro and their biodistribution in an in vivo mouse tumour model. Colocalization of the CDNs at the tumour site in a cancer mouse model was demonstrated, highlighting the potential for CDNs as anti-cancer strategy. Taken together, the results suggest that CDNs could provide a cost-effective alternative to exosomes as an ideal drug nanocarrier.
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http://dx.doi.org/10.1038/s41598-017-14725-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662560PMC
October 2017

Leukocytic Toll-Like Receptor 2 Deficiency Preserves Cardiac Function And Reduces Fibrosis In Sustained Pressure Overload.

Sci Rep 2017 08 23;7(1):9193. Epub 2017 Aug 23.

Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

An involement of Toll-like receptor 2 (TLR2) has been established in cardiac dysfunction after acute myocardial infarction; however, its role in chronic pressure overload is unclear. We sought to evaluate the role of TLR2 in cardiac hypertrophy, fibrosis and dysfunction in sustained pressure overload. We induced pressure overload via transverse aortic constriction (TAC) in TLR2 and wild type (WT) mice, and followed temporal changes over 8 weeks. Despite similar increases in heart weight, left ventricular (LV) ejection fraction (EF) and diastolic function (mitral E/A ratio) were preserved in TLR2 mice but impaired in WT mice following TAC. TAC produced less LV fibrosis in TLR2 mice associated with lower mRNA levels of collagen genes (Col1a1 and Col3a1) and lower protein level of TGFbeta1, compared to WT mice. Following TAC, the influx of macrophages and CD3 T cells into LV was similar between TLR2 and WT mice, whereas levels of cyto/chemokines were lower in the heart and plasma in TLR2 mice. TLR2 bone marrow-derived cells protected against LVEF decline and fibrosis following TAC. Our findings show that leukocytic TLR2 deficiency protects against LV dysfunction and fibrosis probably via a reduction in inflammatory signaling in sustained pressure overload.
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http://dx.doi.org/10.1038/s41598-017-09451-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569043PMC
August 2017

Liposome encapsulated berberine treatment attenuates cardiac dysfunction after myocardial infarction.

J Control Release 2017 02 5;247:127-133. Epub 2017 Jan 5.

Clinical Chemistry and Haematology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Electronic address:

Inflammation is a known mediator of adverse ventricular remodeling after myocardial infarction (MI) that may lead to reduction of ejection fraction and subsequent heart failure. Berberine is a isoquinoline quarternary alkaloid from plants that has been associated with anti-inflammatory, anti-oxidative, and cardioprotective properties. Its poor solubility in aqueous buffers and its short half-life in the circulation upon injection, however, have been hampering the extensive usage of this natural product. We hypothesized that encapsulation of berberine into long circulating liposomes could improve its therapeutic availability and efficacy by protecting cardiac function against MI in vivo. Berberine-loaded liposomes were prepared by ethanol injection and characterized. They contained 0.3mg/mL of the drug and were 0.11μm in diameter. Subsequently they were tested for IL-6 secretion inhibition in RAW 264.7 macrophages and for cardiac function protection against adverse remodeling after MI in C57BL/6J mice. In vitro, free berberine significantly inhibited IL-6 secretion (IC=10.4μM), whereas encapsulated berberine did not as it was not released from the formulation in the time frame of the in vitro study. In vivo, berberine-loaded liposomes significantly preserved the cardiac ejection fraction at day 28 after MI by 64% as compared to control liposomes and free berberine. In conclusion, liposomal encapsulation enhanced the solubility of berberine in buffer and preserves ejection fraction after MI. This shows that delivery of berberine-loaded liposomes significantly improves its therapeutic availability and identifies berberine-loaded liposomes as potential treatment of adverse remodeling after MI.
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http://dx.doi.org/10.1016/j.jconrel.2016.12.042DOI Listing
February 2017

Aberrant Splicing Promotes Proteasomal Degradation of L-type Ca1.2 Calcium Channels by Competitive Binding for Caβ Subunits in Cardiac Hypertrophy.

Sci Rep 2016 10 12;6:35247. Epub 2016 Oct 12.

Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore 117597, Singapore.

Decreased expression and activity of Ca1.2 calcium channels has been reported in pressure overload-induced cardiac hypertrophy and heart failure. However, the underlying mechanisms remain unknown. Here we identified in rodents a splice variant of Ca1.2 channel, named Ca1.2, that contained the pair of mutually exclusive exons 21 and 22. This variant was highly expressed in neonatal hearts. The abundance of this variant was gradually increased by 12.5-folds within 14 days of transverse aortic banding that induced cardiac hypertrophy in adult mouse hearts and was also elevated in left ventricles from patients with dilated cardiomyopathy. Although this variant did not conduct Ca ions, it reduced the cell-surface expression of wild-type Ca1.2 channels and consequently decreased the whole-cell Ca influx via the Ca1.2 channels. In addition, the Ca1.2 variant interacted with Caβ subunits significantly more than wild-type Ca1.2 channels, and competition of Caβ subunits by Ca1.2 consequently enhanced ubiquitination and subsequent proteasomal degradation of the wild-type Ca1.2 channels. Our findings show that the resurgence of a specific neonatal splice variant of Ca1.2 channels in adult heart under stress may contribute to heart failure.
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http://dx.doi.org/10.1038/srep35247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059693PMC
October 2016

Extracellular Vesicle Proteins Associated with Systemic Vascular Events Correlate with Heart Failure: An Observational Study in a Dyspnoea Cohort.

PLoS One 2016 28;11(1):e0148073. Epub 2016 Jan 28.

Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Background: SerpinF2, SerpinG1, CystatinC and CD14 are involved in inflammatory processes and plasma extracellular vesicle (EV) -levels of these proteins have been reported to be associated with systemic vascular events. Evidence is accumulating that inflammatory processes may play a pivotal role both in systemic vascular events and in heart failure. Therefore, we studied the association between plasma extracellular vesicle SerpinF2-, SerpinG1-, CystatinC and CD14-levels and the occurrence of acute heart failure in patients.

Methods And Result: Extracellular vesicle protein levels of SerpinG1, SerpinF2, CystatinC and CD14 were measured in an observational study of 404 subjects presenting with dysponea at the emergency department (4B-cohort). Plasma extracellular vesicles were precipitated in a total extracellular vesicles (TEX)-fraction and in separate LDL- and HDL-subfractions. Extracellular vesicle protein levels were measured with a quantitative immune assay in all 3 precipitates. Out of 404 subjects, 141 (35%) were diagnosed with acutely decompensated heart failure. After correction for confounders (including comorbidities and medications), levels of CD14 in the HDL-fraction (OR 1.53, p = 0.01), SerpinF2 in the TEX-and LDL-fraction (ORs respectively 0.71 and 0.65, p<0.05) and SerpinG1 in the TEX-fraction (OR 1.55, p = 0.004) were statistically significantly related to heart failure. Furthermore, extracellular vesicle CD14- and SerpinF2-levels were significantly higher in heart failure patients with preserved ejection fraction than in those with reduced ejection fraction.

Conclusion: Extracellular vesicle levels of CD14, SerpinG1 and SerpinF2 are associated with the occurrence of heart failure in subjects suspected for acute heart failure, suggesting common underlying pathophysiological mechanisms for heart failure and vascular events.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148073PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731211PMC
July 2016

The diagnostic and prognostic potential of plasma extracellular vesicles for cardiovascular disease.

Expert Rev Mol Diagn 2015 4;15(12):1577-88. Epub 2015 Nov 4.

a Laboratory of Experimental Cardiology , University Medical Center Utrecht , Utrecht , the Netherlands.

Cardiovascular disease (CVD) is the leading cause of death worldwide and its prevalence is expected to rise rapidly worldwide in the coming decades. Atherosclerosis, the syndrome underlying CVD, is a chronic progressive disease of the arteries already present at a young age. Strokes, heart attacks and heart failure are acute CVD events that occur after decades, however, and require timely diagnosis and treatment. Plasma extracellular vesicles (EVs) are microstructures with a lipid bilayer membrane involved in hemostasis, inflammation and injury. Both EV-counts and EV-content are associated with CVD and the identification of plasma EVs is a novel source of blood-based biomarkers with the potential to improve diagnosis and prognosis of CVD. Presented in this review is an overview of the current use of EVs in CVD and a discussion of the need for robust and easy isolation technologies for plasma EV subsets. This is needed to bring this promising field towards clinical application in the patient.
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http://dx.doi.org/10.1586/14737159.2015.1109450DOI Listing
September 2016

Mutations in the D1 domain of von Willebrand factor impair their propeptide-dependent multimerization, intracellular trafficking and secretion.

J Hematol Oncol 2015 Jun 20;8:73. Epub 2015 Jun 20.

Collaborative Innovation Center of Hematology, MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital, Soochow University, Suzhou, 215006, China.

We identified three novel mutations (p.Gly39Arg, p.Lys157Glu, p.Cys379Gly) and one previously known mutation (p.Asp141Asn) in the von Willebrand factor propeptide from three von Willebrand disease patients. All four mutations impaired multimerization of von Willebrand factor, due to reduced oxidoreductase activity of isomeric propeptide. These mutations resulted in the endothelial reticulum retention and impaired basal and stimulated secretions of von Willebrand factor. Our results support that the mutations in the D1 domain lead to defective multimerization, intracellular trafficking, and secretion of von Willebrand factor and result in bleeding of patients.
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http://dx.doi.org/10.1186/s13045-015-0166-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4487848PMC
June 2015

Storage and secretion of naturally occurring von Willebrand factor A domain variants.

Br J Haematol 2014 Nov 8;167(4):529-40. Epub 2014 Aug 8.

Einthoven Laboratory for Experimental Vascular Medicine, Department of Thrombosis and Hemostasis, Leiden University Medical Centre, Leiden, The Netherlands.

Von Willebrand disease (VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1-A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with plasmids encoding full-length wild-type VWF or mutant VWF. Six mutations in the A1-A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo-Weibel-Palade bodies (WPB). In addition, two mutations also showed reduced numbers of pseudo-WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion.
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http://dx.doi.org/10.1111/bjh.13074DOI Listing
November 2014

Serum extracellular vesicle protein levels are associated with acute coronary syndrome.

Eur Heart J Acute Cardiovasc Care 2013 Mar;2(1):53-60

University Medical Center Utrecht, Utrecht, The Netherlands.

Aims: Biomarkers are essential in the early detection of acute coronary syndromes (ACS). Serum extracellular vesicles are small vesicles in the plasma containing protein and RNA and have been shown to be involved in ACS-related processes like apoptosis and coagulation. Therefore, we hypothesized that serum extracellular vesicle protein levels are associated with ACS.

Methods And Results: Three serum extracellular vesicle proteins potentially associated with ACS were identified with differential Q-proteomics and were evaluated in 471 frozen serum samples of ACS-suspected patients presenting to the emergency department (30% of whom had an ACS). Protein levels were measured after vesicle isolation using ExoQuick. Mean serum extracellular vesicle concentration of the different proteins was compared between ACS and non-ACS patients. Selected proteins were tested in a univariate logistic regression model, as well as in a multivariate model to adjust for cardiovascular risk factors. A separate analysis was performed in men and women. In the multivariate logistic regression analysis, polygenic immunoglobulin receptor, (pIgR; OR 1.630, p=0.026), cystatin C (OR 1.641, p=0.021), and complement factor C5a (C5a, OR 1.495, p=0.025) were significantly associated with ACS, while total vesicle protein concentration was borderline significant. The association of the individual proteins with ACS was markedly stronger in men.

Conclusions: These data show that serum extracellular vesicle pIgR, cystatin C, and C5a concentrations are independently associated with ACS and that there are pronounced gender differences. These observations should be validated in a large, prospective study to assess the potential role of vesicle content in the evaluation of patients suspected of having an ACS.
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http://dx.doi.org/10.1177/2048872612471212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760575PMC
March 2013