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Growth factor stimulation of cardiomyocytes induces changes in the transcriptional contents of secreted exosomes
Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine.
Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine. Department of Cardiology, Heart Centre, Umeå University, Umeå, Sweden.ORCID iD: 0000-0002-3822-0725
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Institutionen för skoglig genetik och växtfysiologi, Sveriges lantbruks universitet.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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2013 (English)In: Journal of Extracellular Vesicles, E-ISSN 2001-3078, article id 20167Article in journal (Refereed) Published
Abstract [en]

Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in targets cells. We recently reported that cultured cardiomyocytes are able to release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the transcriptional content of the released exosomes.

Exosomes were isolated from media collected from cultured cardiomyocytes (HL-1) with or without growth factor treatment (TGF-β2 and PDGF-BB), with a series of differential centrifugations, including preparative ultracentrifugation and separation with a sucrose gradient. The exosomes were characterized with dynamic light scattering (DLS), electron microscopy (EM) and Western blot and analyzed with Illumina whole genome microarray gene expression.

The exosomes were rounded in shape and had an average size of 50–90 nm in diameter with no difference between treatment groups. Analysis of the mRNA content in repeated experiments conclusively revealed 505 transcripts in the control group, 562 in the TGF-β2-treated group and 300 in the PDGF-BB-treated group. Common transcripts (217) were found in all 3 groups.

We show that the mode of stimulation of parental cells affects the characteristics of exosomes released. Hence, there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated, or not stimulated, with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system.

Place, publisher, year, edition, pages
Co-Action Publishing , 2013. article id 20167
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-59474DOI: 10.3402/jev.v2i0.20167Scopus ID: 2-s2.0-84887518558OAI: oai:DiVA.org:umu-59474DiVA, id: diva2:552445
Note

Originally included in thesis in manuscript form with title: Characterisation of exosomes derived from cultured cardiomyocytes treated with different growth factors

Available from: 2012-09-14 Created: 2012-09-14 Last updated: 2024-01-17Bibliographically approved
In thesis
1. Cardiac hypertrophy: transcription patterns, hypertrophic progression and extracellular signalling
Open this publication in new window or tab >>Cardiac hypertrophy: transcription patterns, hypertrophic progression and extracellular signalling
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Hjärthypertrofi : transkriptionsmönster, hypertrofisk progression och extracellulär signalering
Abstract [en]

Background: The aim of this thesis was to study transcription patterns and extracellular signalling of the hypertrophic heart to better understand the mechanisms initiating, controlling and maintaining cardiac hypertrophy.

Cardiac hypertrophy is a risk factor for cardiovascular morbidity and mortality. Hypertrophy of the myocardium is a state, independent of underlying disease, where the myocardium strives to compensate for an increased workload. This remodelling of the heart includes physiological changes induced by a changed gene expression, alteration of the extracellular matrix and diverse cell-to-cell signalling.

Shedding microvesicles and exosomes are membrane released vesicles derived from the plasma membrane, which can mediate messages between cells and induce various cell-related processes in target cells.

Methods and materials: Two different microarray studies on different materials were performed. In the first study, cardiac myectomies from 8 patients with hypertrophic obstructive cardiomyopathy (HOCM) and 5 controls without cardiac disease were used. In the second study, myocardial tissue from 6 aorta ligated and 6 sham operated (controls) rats at three different time points (1, 6 and 42 days post-surgically) were analysed. To reveal differences in gene expression the materials were analyzed with Illumina whole genome microarray and multivariate data analysis (PCA and OPLS-DA).

Cultured cardiomyocytes (HL-1) were incubated with and without growth factors (TGF-β2 or PDGF BB). Microvesicles and exosomes were collected and isolated after differential centrifugations and ultracentrifugations of the cell culture medium. The microvesicles and exosomes were characterized with dynamic light scattering (DLS), flow cytometry, western blot, electron microscopy and Illumina whole genome microarray.

Results: The two different microarray studies revealed differentially expressed gene transcripts and groups of transcripts. When comparing HOCM patients to controls significant down-regulation of the MYH6 gene transcript and two immediate early genes (IEGs, EGR1 and FOS), as well as significant up-regulation of the ACE2, JAK2 and HDAC5 gene transcripts were found. In the rat model, 5 gene groups showed interesting clustering after multivariate data analysis (OPLS-DA) associated with the hypertrophic development: “Atherosclerosis”, “ECM and adhesion molecules”, “Fatty acid metabolism”, “Glucose metabolism” and “Mitochondria”.

The shedding microvesicles were rounded vesicles, 40-300 nm in size and surrounded by a bilayered membrane. Chromosomal DNA sequences were identified in the microvesicles. The microvesicles could be taken up by fibroblasts resulting in an altered gene expression in the fibroblasts. The exosomes from cultured cardiomyocytes (incubated with TGF-β2 or PDGF BB) had an average diameter of 50-80 nm, similar to the unstimulated control exosomes. A large, for all cardiomyocyte derived exosomes, common pool of mRNA seems stable and a smaller pool varied in mRNA content according to treatment of the cardiomyocyte. Of the common mRNA about 14% were ribosomal, 14% were of unknown locus and 5% connected to the function of the mitochondria.

Conclusions: The microarray studies showed that transcriptional regulation at a stable stage of the hypertrophic development is a balance of pro and anti hypertrophic mechanisms and that diverse gene groups are differently regulated at different time points in the hypertrophic progression.

OPLS-DA is a very useful and powerful tool when analyzing gene expression data, especially in finding clusters of gene groups not seen with traditional statistics.

The extracellular vesicle studies suggests that microvesicles and exosomes released from cardiomyocytes contain DNA and can be involved in events in target cells by facilitating an array of processes including gene expression changes. Different treatment of the cardiomyocyte influence the content of the exosome produced, indicating that the signal function of the exosome might vary according to the state of the cardiomyocyte.

Abstract [sv]

Bakgrund: Syftet med den här avhandlingen var att studera transkriptions-mönster och extracellulär signalering vid hjärthypertrofi för att bättre förstå de mekanismer som startar, styr och underhåller tillväxten. Hjärthypertrofi, onormal tillväxt av hjärtmuskeln, är en riskfaktor för andra hjärt-kärlsjukdomar och dödlighet. Hypertrofi av hjärtmuskeln är ett tillstånd, oberoende av bakomliggande sjukdom, där hjärtmuskeln strävar efter att kompensera för ökad arbetsbelastning. Denna omställning av hjärtat innefattar fysiologiska förändringar orsakade av ett förändrat genuttryck, modifiering av miljön utanför cellen och ändrad cell-till-cell signalering.

Mikrovesiklar och exosomer är små membranomslutna bubblor som frisätts från cellmembranet, ut i cellens omgivning. De kan förmedla budskap mellan celler och påverka olika processer i målceller.

Metoder och material: Avhandlingen innefattar två olika microarraystudier på olika material. I den första studien användes hjärtbiopsier från 8 patienter med hypertrofisk obstruktiv kardiomyopati (HOCM) och 5 kontroller utan hjärtsjukdom. I det andra projektet användes hjärtvävnad från 6 aortaligerade och 6 skenopererade (kontroller) råttor vid tre olika tidpunkter (1, 6 och 42 dagar efter kirurgiskt ingrepp). För att påvisa skillnader i genuttryck analyserades proverna med Illumina helgenom microarray och multivariat dataanalys.

Avhandlingens andra del innehåller två studier om mikrovesiklar och exosomer. Odlade hjärtmuskelceller (HL-1) stimulerades med tillväxt-faktorer (TGF-β2 eller PDGF BB) och ostimulerade celler användes som kontroll. Mikrovesiklar och exosomer renades fram med centrifugeringar och ultracentrifugering av cellodlingsmediet för att sedan karakteriseras med olika metoder för att studera storlek, ytmarkörer och innehåll. Illumina helgenom microarray användes för att studera microvesiklarnas och exosomernas mRNA innehåll.

Resultat: I de två olika microarraystudierna hittades gentranskript och grupper av gentranskript som skiljde sig mellan kontroller och den hypertrofa hjärtvävnaden. När HOCM patientproverna jämfördes med kontroller hittades nedreglering av MYH6, EGR1 och FOS samt uppreglering av ACE2, JAK2 och HDAC5. Efter multivariat dataanalys av materialet från råtta, hittades 5 grupper av gentranskript med intressanta mönster som kunde kopplas till den hypertrofiska utvecklingen av hjärtmuskeln: "Ateroskleros", "ECM och adhesionsmolekyler", "Fettsyrametabolism", "Glukosmetabolis-men" och "Mitokondrien".

Mikrovesiklarna hade en diameter på 40-300 nm och innehöll kromosomala DNA-sekvenser. När mikrovesiklarna överfördes till en annan celltyp (fibroblaster) resulterade det i ett förändrat genuttryck i fibroblasterna. Exosomer från hjärtmuskelcellerna som odlats med eller utan tillväxtfaktor hade en diameter på 50-80 nm. En stor pool av olika gentranskript var gemensam för alla exosomer oavsett stimulering eller ej. En mindre pool av gentranskript varierade i innehåll mellan de stimulerade och ostimulerade hjärtmuskelcellerna. I den gemensamma gentranskript poolen var ca 14 % ribosomala, ca 14 % var okända och ca 5 % var associerade till mitokondrien och dess funktion.

Slutsats: Microarraystudierna visade att transkriptionsreglering i ett stabilt skede av hypertrofiutvecklingen är en balans mellan pro- och anti-hypertrofiska mekanismer och att olika gengrupper var olika reglerade vid olika tidpunkter i hjärtmuskeltillväxten.

OPLS-DA är ett mycket användbart och kraftfullt verktyg när man analyserar genexpressionsdata, särskilt för att hitta grupper av gen-transkript som är svåra att upptäcka med traditionell statistik.

Microvesikel- och exosomstudierna visade att mikrovesiklar och exosomer som frisätts från hjärtmuskelceller innehåller både DNA och RNA och kan vara inblandade i händelserna i målceller genom att underlätta en rad processer, inklusive ändringar av genuttryck. Olika stimulering av hjärtmuskelcellen kan påverka innehållet i exosomernas som produceras, vilket indikerar att exosomernas signalfunktion kan variera beroende på hjärtmuskelcellens tillstånd.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2012. p. 64
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1516
Keywords
Cardiac hypertrophy, HCM, gene expression, OPLS-DA, multivariate dataanalysis, cardiomyocytes, exosomes, microvesicles
National Category
Cardiac and Cardiovascular Systems
Research subject
Medicine
Identifiers
urn:nbn:se:umu:diva-59470 (URN)978-91-7459-468-3 (ISBN)
Public defence
2012-10-05, Sal D, 9 trappor, byggnad 1D, NUS, Norrlands Universitetssjukhus, 90185 Umeå, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-09-14 Created: 2012-09-14 Last updated: 2022-12-13Bibliographically approved

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Gennebäck, NinaHellman, UrbanMalm, LinusLarsson, GöranWaldenström, AndersMörner, Stellan

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