Ischemia challenged epicardial adipose tissue stem cells-derived extracellular vesicles alter the gene expression of cardiac fibroblasts to cardiomyocyte like phenotype

Published:October 20, 2022DOI:


      The present study hypothesizes that the ischemic insults activate epicardial adipose tissue-derived stem cells (EATDS) to secrete extracellular vesicles (EVs) packed with regenerative mediators to alter the gene expression in cardiac fibroblasts (CF). EATDS and CF were isolated from hyperlipidemic microswine and EVs were harvested from control, simulated ischemia (ISC) and ischemia-reperfusion (ISC/R) groups. The in vitro interaction between ISC–EVs and CF resulted in the upregulation of cardiomyocyte-specific transcription factors including GATA4, Nkx2.5, IRX4, and TBX5 in CF and the healing marker αSMA and the downregulation of fibroblast biomarkers such as vimentin, FSP1, and podoplanin and the cardiac biomarkers such as troponin-I and connexin-43. These results suggest a cardiomyocyte-like phenotype as confirmed by immunostaining and Western blot. The LC-MS/MS analysis of ISC–EVs LGALS1, PRDX2, and CCL2 to be the potent protein mediators which are intimately involved in versatile regenerative processes and connected with a diverse array of regenerative genes. Moreover, the LGALS1+, PRDX2+, and CCL2+ EATDS phenotypes were deciphered at single cell resolution revealing corresponding sub-populations with superior healing potential. Overall, the findings unveiled the healing potential of EATDS-derived EVs and sub-populations of regenerative EATDS promising novel translational opportunities in improved cardiac healing following ischemic injury.



      BCA (Bicinchoninic acid), CF (Cardiac fibroblasts), DAPI (4',6-diamidino-2-phenylindole), EATDS (Epicardial adipose tissue-derived stem cells), EVs (Extracellular vesicles), ISC (Ischemia), ISC/R (Ischemia-reperfusion), MI (myocardial infarction), MSC (Mesenchymal stem cells), PBS (Phosphate buffered saline)
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        • Cui B
        • Zheng Y
        • Sun L
        • et al.
        Heart regeneration in adult mammals after myocardial damage.
        Acta Cardiol Sin. 2018; 34: 115-123
        • Agrawal DK.
        Commentary: unraveling the mystery of transient innate capacity for neonatal heart regeneration following injury.
        J Thorac Cardiovasc Surg. 2021; (Published online September)S0022522321012745
        • Agrawal DK
        • Thankam FG.
        Commentary: evidence-based human stem cell therapy for myocardial healing: Miles to go.
        JTCVS Open. 2021; (Published online July)S2666273621001686
        • Giacca M.
        Cardiac regeneration after myocardial infarction: an approachable goal.
        Curr Cardiol Rep. 2020; 22
        • Cahill TJ
        • Choudhury RP
        • Riley PR.
        Heart regeneration and repair after myocardial infarction: translational opportunities for novel therapeutics.
        Nat Rev Drug Discov. 2017; 16: 699-717
        • Guo X
        • Bai Y
        • Zhang L
        • et al.
        Cardiomyocyte differentiation of mesenchymal stem cells from bone marrow: new regulators and its implications.
        Stem Cell Res Ther. 2018; 9: 44
        • Gao Q
        • Guo M
        • Jiang X
        • et al.
        A cocktail method for promoting cardiomyocyte differentiation from bone marrow-derived Mesenchymal stem cells.
        Stem Cells Int. 2014; (2014)e162024
        • Pittenger MF
        • Martin BJ.
        Mesenchymal stem cells and their potential as cardiac therapeutics.
        Circ Res. 2004; 95: 9-20
        • Guo Y
        • Yu Y
        • Hu S
        • et al.
        The therapeutic potential of mesenchymal stem cells for cardiovascular diseases.
        Cell Death Dis. 2020; 11: 1-10
        • Chen Y
        • Yang Z
        • Zhao ZA
        • Shen Z.
        Direct reprogramming of fibroblasts into cardiomyocytes.
        Stem Cell Res Ther. 2017; 8: 118
        • Thankam FG
        • Agrawal DK.
        Single cell genomics identifies unique cardioprotective phenotype of stem cells derived from epicardial adipose tissue under ischemia.
        Stem Cell Rev Rep. 2021; 18 (Published online October)
        • Thankam FG
        • Chandra I
        • Diaz C
        • et al.
        Matrix regeneration proteins in the hypoxia-triggered exosomes of shoulder tenocytes and adipose-derived mesenchymal stem cells.
        Mol Cell Biochem. 2019; (Published online December 3)
        • Thankam FG
        • Ayoub JG
        • Ahmed MMR
        • et al.
        Association of hypoxia and mitochondrial damage associated molecular patterns in the pathogenesis of vein graft failure: a pilot study.
        Transl Res. 2020; (Published online August 28)
        • Rai V
        • Agrawal DK.
        Immunomodulation of IL-33 and IL-37 with vitamin D in the neointima of coronary artery: a comparative study between balloon angioplasty and stent in hyperlipidemic microswine.
        Int J Mol Sci. 2021; 22: 8824
        • Thankam FG
        • Chandra IS
        • Kovilam AN
        • et al.
        Amplification of mitochondrial activity in the healing response following rotator cuff tendon injury.
        Sci Rep. 2018; 8: 1-14
        • Théry C
        • Witwer KW
        • Aikawa E
        • et al.
        Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.
        J Extracell Vesicles. 2018; 71535750
        • Thankam FG
        • Agrawal DK.
        Hypoxia-driven secretion of extracellular matrix proteins in the exosomes reflects the asymptomatic pathology of rotator cuff tendinopathies.
        Can J Physiol Pharmacol. 2020; 15 (Published online August)
        • Thankam FG
        • Larsen NK
        • Varghese A
        • et al.
        Biomarkers and heterogeneous fibroblast phenotype associated with incisional hernia.
        Mol Cell Biochem. 2021; 476: 3353-3363
        • Rappsilber J
        • Mann M
        • Ishihama Y.
        Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.
        Nat Protoc. 2007; 2: 1896-1906
        • Thankam FG
        • Agrawal DK.
        Hypoxia-driven secretion of extracellular matrix proteins in the exosomes reflects the asymptomatic pathology of rotator cuff tendinopathies.
        Can J Physiol Pharmacol. 2020; 15 (Published online Augustcjpp-2020-0314)
        • Thankam FG
        • Boosani CS
        • Dilisio MF
        • et al.
        MicroRNAs associated with shoulder tendon matrisome disorganization in glenohumeral arthritis.
        PloS One. 2016; 11e0168077
        • Caruso Bavisotto C
        • Scalia F
        • Marino Gammazza A
        • et al.
        Extracellular vesicle-mediated cell–cell communication in the nervous system: focus on neurological diseases.
        Int J Mol Sci. 2019; 20: 434
        • Cherian S
        • Lopaschuk GD
        • Carvalho E.
        Cellular cross-talk between epicardial adipose tissue and myocardium in relation to the pathogenesis of cardiovascular disease.
        Am J Physiol-Endocrinol Metab. 2012; 303: E937-E949
        • Gaborit B
        • Venteclef N
        • Ancel P
        • et al.
        Human epicardial adipose tissue has a specific transcriptomic signature depending on its anatomical peri-atrial, peri-ventricular, or peri-coronary location.
        Cardiovasc Res. 2015; 108: 62-73
        • Gruzdeva O
        • Uchasova E
        • Dyleva Y
        • et al.
        Relationships between epicardial adipose tissue thickness and adipo-fibrokine indicator profiles post-myocardial infarction.
        Cardiovasc Diabetol. 2018; 17: 40
        • Lambert C
        • Arderiu G
        • Bejar MT
        • et al.
        Stem cells from human cardiac adipose tissue depots show different gene expression and functional capacities.
        Stem Cell Res Ther. 2019; 10: 361
        • Wystrychowski W
        • Patlolla B
        • Zhuge Y
        • et al.
        Multipotency and cardiomyogenic potential of human adipose-derived stem cells from epicardium, pericardium, and omentum.
        Stem Cell Res Ther. 2016; 7
        • Alonso-Alonso ML
        • García-Posadas L
        • Diebold Y
        Extracellular vesicles from human adipose-derived mesenchymal stem cells: a review of common cargos.
        Stem Cell Rev Rep. 2021; (Published online April 26)
        • Wu Q
        • Wang J
        • Tan WLW
        • et al.
        Extracellular vesicles from human embryonic stem cell-derived cardiovascular progenitor cells promote cardiac infarct healing through reducing cardiomyocyte death and promoting angiogenesis.
        Cell Death Dis. 2020; 11: 1-16
        • Chen W
        • Bian W
        • Zhou Y
        • Zhang J.
        Cardiac fibroblasts and myocardial regeneration.
        Front Bioeng Biotechnol. 2021; 9599928
        • Hara H
        • Takeda N
        • Komuro I.
        Pathophysiology and therapeutic potential of cardiac fibrosis.
        Inflamm Regen. 2017; 37: 13
        • Talman V
        • Ruskoaho H.
        Cardiac fibrosis in myocardial infarction—from repair and remodeling to regeneration.
        Cell Tissue Res. 2016; 365: 563-581
        • Gomes RN
        • Manuel F
        • Nascimento DS.
        The bright side of fibroblasts: molecular signature and regenerative cues in major organs.
        Npj Regen Med. 2021; 6: 1-12
        • Wang Z
        • Cui M
        • Shah AM
        • et al.
        Cell-type-specific gene regulatory networks underlying murine neonatal heart regeneration at single-cell resolution.
        Cell Rep. 2020; 33108472
        • Porrello ER
        • Mahmoud AI
        • Simpson E
        • et al.
        Transient regenerative potential of the neonatal mouse heart.
        Science. 2011; 331: 1078-1080
        • Quaife-Ryan GA
        • Sim CB
        • Ziemann M
        • et al.
        Multicellular transcriptional analysis of mammalian heart regeneration.
        Circulation. 2017; 136: 1123-1139
        • Furtado MB
        • Costa MW
        • Pranoto EA
        • et al.
        Cardiogenic genes expressed in cardiac fibroblasts contribute to heart development and repair.
        Circ Res. 2014; 114: 1422-1434
        • Zhang Z
        • Zhang AD
        • Kim LJ
        • Nam YJ.
        Ensuring expression of four core cardiogenic transcription factors enhances cardiac reprogramming.
        Sci Rep. 2019; 9: 6362
        • Dal-Pra S
        • Hodgkinson CP
        • Dzau VJ.
        Induced cardiomyocyte maturation: Cardiac transcription factors are necessary but not sufficient.
        PLoS ONE. 2019; 14
        • Saludas L
        • Oliveira CC
        • Roncal C
        • et al.
        Extracellular vesicle-based therapeutics for heart repair.
        Nanomaterials. 2021; 11: 570
        • Fu S
        • Zhang Y
        • Li Y
        • et al.
        Extracellular vesicles in cardiovascular diseases.
        Cell Death Discov. 2020; 6: 1-9
        • Gao L
        • Wang L
        • Wei Y
        • et al.
        Exosomes secreted by hiPSC-derived cardiac cells improve recovery from myocardial infarction in swine.
        Sci Transl Med. 2020; 12
        • Martínez MS
        • García A
        • Luzardo E
        • et al.
        Energetic metabolism in cardiomyocytes: molecular basis of heart ischemia and arrhythmogenesis.
        Vessel Plus. 2017; 1: 130-141
        • Pluijmert NJ
        • Atsma DE
        • Quax PHA.
        Post-ischemic myocardial inflammatory response: a complex and dynamic process susceptible to immunomodulatory therapies.
        Front Cardiovasc Med. 2021; 8: 207
        • Seropian IM
        • Cerliani JP
        • Toldo S
        • et al.
        Galectin-1 controls cardiac inflammation and ventricular remodeling during acute myocardial infarction.
        Am J Pathol. 2013; 182: 29-40
        • Al-Salam S
        • Hashmi S.
        Galectin-1 in early acute myocardial infarction.
        PLOS ONE. 2014; 9: e86994
        • Li H
        • Yang H
        • Wang D
        • Zhang L
        • Ma T.
        Peroxiredoxin2 (Prdx2) reduces oxidative stress and apoptosis of myocardial cells induced by acute myocardial infarction by inhibiting the TLR4/Nuclear Factor kappa B (NF-κB) signaling pathway.
        Med Sci Monit Int Med J Exp Clin Res. 2020; 26 (1-e926281-11)e926281
        • Jin X
        • Chen C
        • Li D
        • et al.
        PRDX2 in myocyte hypertrophy and survival is mediated by TLR4 in acute infarcted myocardium.
        Sci Rep. 2017; 7: 6970
        • Won H
        • Lim S
        • Jang M
        • et al.
        Peroxiredoxin-2 upregulated by NF-κB attenuates oxidative stress during the differentiation of muscle-derived C2C12 cells.
        Antioxid Redox Signal. 2012; 16: 245-261
        • Wang S
        • Chen Z
        • Zhu S
        • et al.
        PRDX2 protects against oxidative stress induced by H. pylori and promotes resistance to cisplatin in gastric cancer.
        Redox Biol. 2020; 28101319
        • Li J
        • Wang C
        • Wang W
        • et al.
        prdx2 protects against atherosclerosis by regulating the phenotype and function of the vascular smooth muscle cell.
        Front Cardiovasc Med. 2021; 8: 128
        • Mullen L
        • Hanschmann EM
        • Lillig CH
        • et al.
        Cysteine oxidation targets peroxiredoxins 1 and 2 for exosomal release through a novel mechanism of redox-dependent secretion.
        Mol Med. 2015; 21: 98-108
        • Frangogiannis NG
        • Smith CW
        • Entman ML.
        The inflammatory response in myocardial infarction.
        Cardiovasc Res. 2002; 53: 31-47
        • Hayashidani S
        • Tsutsui H
        • Shiomi T
        • et al.
        Anti-monocyte chemoattractant protein-1 gene therapy attenuates left ventricular remodeling and failure after experimental myocardial infarction.
        Circulation. 2003; 108: 2134-2140
        • Morimoto H
        • Takahashi M
        • Izawa A
        • et al.
        cardiac overexpression of monocyte chemoattractant protein-1 in transgenic mice prevents cardiac dysfunction and remodeling after myocardial infarction.
        Circ Res. 2006; 99: 891-899
        • Lee S
        • Kim OJ
        • Lee KO
        • et al.
        Enhancing the therapeutic potential of CCL2-overexpressing mesenchymal stem cells in acute stroke.
        Int J Mol Sci. 2020; 21: 7795
        • Lee HK
        • Kim HS
        • Kim JS
        • et al.
        CCL2 deficient mesenchymal stem cells fail to establish long-lasting contact with T cells and no longer ameliorate lupus symptoms.
        Sci Rep. 2017; 7: 41258
        • Hasegawa Y
        • Tang D
        • Takahashi N
        • et al.
        CCL2 enhances pluripotency of human induced pluripotent stem cells by activating hypoxia related genes.
        Sci Rep. 2014; 4: 5228
        • Whelan DS
        • Caplice NM
        • Clover AJP.
        Mesenchymal stromal cell derived CCL2 is required for accelerated wound healing.
        Sci Rep. 2020; 10: 2642