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Thromb Haemost 2017; 117(03): 529-542
DOI: 10.1160/TH16-04-0322
Cellular Haemostasis and Platelets
Schattauer GmbH

Hyperglycaemia suppresses microRNA expression in platelets to increase P2RY12 and SELP levels in type 2 diabetes mellitus

Zsolt Fejes
1   Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
,
Szilárd Póliska
2   Department of Biochemistry and Molecular Biology, Genomic Medicine and Bioinformatics Core Facility, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
,
Zsolt Czimmerer
2   Department of Biochemistry and Molecular Biology, Genomic Medicine and Bioinformatics Core Facility, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
,
Miklós Káplár
3   Institute of Internal Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
,
András Penyige
4   Department of Human Genetics, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
5   Faculty of Pharmacy, University of Debrecen, Debrecen, Hungary
,
Gabriella Gál Szabó
1   Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
,
Ildikó Beke Debreceni
1   Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
,
Satya P. Kunapuli
6   Department of Physiology and Sol Sherry Thrombosis Center, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
,
János Kappelmayer
1   Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
,
Béla Nagy Jr
1   Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
› Author Affiliations
Further Information

Publication History

Received: 23 April 2016

Accepted after major revision: 26 November 2016

Publication Date:
22 November 2017 (online)

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Summary

Megakaryocyte (MK)-derived miRNAs have been detected in platelets. Here, we analysed the expression of platelet and circulating miR-223, miR-26b, miR-126 and miR-140 that might be altered with their target mRNAs in type 2 diabetes mellitus (DM2). MiRNAs were isolated from leukocyte-depleted platelets and plasma samples obtained from 28 obese DM2, 19 non-DM obese and 23 healthy individuals. The effect of hyperglycaemia on miRNAs was also evaluated in MKs using MEG-01 and K562 cells under hyperglycaemic conditions after 8 hours up to four weeks. Quantitation of mature miRNA, pre-miRNAs and target mRNA levels (P2RY12 and SELP) were measured by RT-qPCR. To prove the association of miR-26b and miR-140 with SELP (P-selectin) mRNA level, overexpression or inhibition of these miRNAs in MEG-01 MKs was performed using mimics or anti-miRNAs, respectively. The contribution of calpain substrate Dicer to modulation of miRNAs was studied by calpain inhibition. Platelet activation was evaluated via surface P-selectin by flow cytometry. Mature and pre-forms of investigated miRNAs were significantly reduced in DM2, and platelet P2RY12 and SELP mRNA levels were elevated by two-fold at increased platelet activation compared to controls. Significantly blunted miRNA expressions were observed by hyperglycaemia in MEG-01 and K562-MK cells versus baseline values, while the manipulation of miR-26b and miR-140 expression affected SELP mRNA level. Calpeptin pretreatment restored miRNA levels in hyperglycaemic MKs. Overall, miR-223, miR-26b, miR-126 and miR-140 are expressed at a lower level in platelets and MKs in DM2 causing upregulation of P2RY12 and SELP mRNAs that may contribute to adverse platelet function.

Supplementary Material to this article is available online at www.thrombosis-online.com.

Keywords

Platelet activation - microRNA - megakaryocyte - diabetes mellitus - hyperglycaemia

 

  • References

  • 1 Ferroni P, Basili S, Falco A. et al. Platelet activation in type 2 diabetes mellitus. J Thromb Haemost 2004; 02: 1282-1291.
    CrossrefPubMedSearch in Google Scholar
  • 2 Nagy Jr B, Csongrádi E, Bhattoa HP. et al. Investigation of Thr715Pro P-selectin gene polymorphism and soluble P-selectin levels in type 2 diabetes mellitus. Thromb Haemost 2007; 98: 186-191.
    Thieme ConnectPubMedSearch in Google Scholar
  • 3 Khaw KT, Wareham N, Bingham S. et al. Association of hemoglobin A1c with cardiovascular disease and mortality in adults: the European prospective investigation into cancer in Norfolk. Ann Intern Med 2004; 141: 413-420.
    CrossrefPubMedSearch in Google Scholar
  • 4 Lemkes BA, Hermanides J, Devries JH. et al. Hyperglycaemia: a prothrombotic factor?. J Thromb Haemost 2010; 08: 1663-1669.
    CrossrefPubMedSearch in Google Scholar
  • 5 Yngen M, Ostenson CG, Li N. et al. Acute hyperglycaemia increases soluble P-selectin in male patients with mild diabetes mellitus. Blood Coagul Fibrinolysis 2001; 12: 109-116.
    CrossrefPubMedSearch in Google Scholar
  • 6 Holman RR, Paul SK, Bethel MA. et al. 10-year follow-up of intensive glucose control in type 2 diabetes. N Engl J Med 2008; 359: 1577-1589.
    CrossrefPubMedSearch in Google Scholar
  • 7 Tschoepe D. The activated megakaryocyte-platelet-system in vascular disease: focus on diabetes. Semin Thromb Hemost 1995; 21: 152-160.
    Thieme ConnectPubMedSearch in Google Scholar
  • 8 Fadini GP, Ferraro F, Quaini F. et al. Concise review: diabetes, the bone marrow niche, and impaired vascular regeneration. Stem Cells Transl Med 2014; 03: 949-957.
    CrossrefPubMedSearch in Google Scholar
  • 9 Olivieri F, Bonafè M, Spazzafumo L. et al. Age- and glycaemia-related miR-126-3p levels in plasma and endothelial cells. Aging 2014; 06: 771-787.
    CrossrefPubMedSearch in Google Scholar
  • 10 Landry P, Plante I, Ouellet DL. et al. Existence of a microRNA pathway in anucleate platelets. Nat Struct Mol Biol 2009; 16: 961-966.
    CrossrefPubMedSearch in Google Scholar
  • 11 Rondina MT, Weyrich AS. Regulation of the genetic code in megakaryocytes and platelets. J Thromb Haemost 2015; 13 (Suppl. 01) S26-32.
    CrossrefPubMedSearch in Google Scholar
  • 12 Pan Y, Liang H, Liu H. et al. Platelet-secreted microRNA-223 promotes endothelial cell apoptosis induced by advanced glycation end products via targeting the insulin-like growth factor 1 receptor. J Immunol 2014; 192: 437-446.
    CrossrefPubMedSearch in Google Scholar
  • 13 Teruel-Montoya R, Kong X, Abraham S. et al. MicroRNA expression differences in human hematopoietic cell lineages enable regulated transgene expression. PLoS One 2014; 09: e102259.
    CrossrefPubMedSearch in Google Scholar
  • 14 Nagalla S, Shaw C, Kong X. et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood 2011; 117: 5189-5197.
    CrossrefPubMedSearch in Google Scholar
  • 15 Zimmerman GA, Weyrich AS. Signal-dependent protein synthesis by activated platelets: new pathways to altered phenotype and function. Arterioscler Thromb Vasc Biol 2008; 28: s17-24.
    CrossrefPubMedSearch in Google Scholar
  • 16 Liverani E, Kilpatrick LE, Tsygankov AY. et al. The role of P2Y12 receptor and activated platelets during inflammation. Curr Drug Targets 2014; 15: 720-728.
    CrossrefPubMedSearch in Google Scholar
  • 17 Matsuno H, Tokuda H, Ishisaki A. et al. P2Y12 receptors play a significant role in the development of platelet microaggregation in patients with diabetes. J Clin Endocrinol Metab 2005; 90: 920-927.
    CrossrefPubMedSearch in Google Scholar
  • 18 Sudic D, Razmara M, Forslund M. et al. High glucose levels enhance platelet activation: involvement of multiple mechanisms. Br J Haematol 2006; 133: 315-322.
    CrossrefPubMedSearch in Google Scholar
  • 19 Nagy Jr B, Jin J, Ashby B. et al. Contribution of the P2Y12 receptor-mediated pathway to platelet hyperreactivity in hypercholesterolaemia. J Thromb Haemost 2011; 09: 810-819.
    CrossrefPubMedSearch in Google Scholar
  • 20 Kondkar AA, Bray MS, Leal SM. et al. VAMP8/endobrevin is overexpressed in hyperreactive human platelets: suggested role for platelet microRNA. J Thromb Haemost 2010; 08: 369-378.
    CrossrefPubMedSearch in Google Scholar
  • 21 Duan X, Zhan Q, Song B. et al. Detection of platelet microRNA expression in patients with diabetes mellitus with or without ischemic stroke. J Diabetes Complications 2014; 28: 705-710.
    CrossrefPubMedSearch in Google Scholar
  • 22 Elgheznawy A, Shi L, Hu J. et al. Dicer cleavage by calpain determines platelet microRNA levels and function in diabetes. Circ Res 2015; 117: 157-165.
    CrossrefPubMedSearch in Google Scholar
  • 23 Stenberg PE, McEver RP, Shuman MA. et al. A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol 1985; 101: 880-886.
    CrossrefPubMedSearch in Google Scholar
  • 24 Kappelmayer J, Nagy Jr B, Miszti-Blasius K. et al. The emerging value of P-selectin as a disease marker. Clin Chem Lab Med 2004; 42: 475-486.
    PubMedSearch in Google Scholar
  • 25 Plé H, Landry P, Benham A. et al. The repertoire and features of human platelet microRNAs. PLoS One 2012; 07: e50746.
    CrossrefPubMedSearch in Google Scholar
  • 26 Czimmerer Z, Hulvely J, Simandi Z. et al. A versatile method to design stem-loop primer-based quantitative PCR assays for detecting small regulatory RNA molecules. PLoS One 2013; 08: e55168.
    CrossrefPubMedSearch in Google Scholar
  • 27 Westerweel PE, Teraa M, Rafii S. et al. Impaired endothelial progenitor cell mobilisation and dysfunctional bone marrow stroma in diabetes mellitus. PLoS One 2013; 08: e60357.
    CrossrefPubMedSearch in Google Scholar
  • 28 Duncan MT, Shin S, Wu JJ. et al. Dynamic transcription factor activity profiles reveal key regulatory interactions during megakaryocytic and erythroid differentiation. Biotechnol Bioeng 2014; 111: 2082-2094.
    CrossrefPubMedSearch in Google Scholar
  • 29 Dobiásová M. Atherogenic index of plasma [log(triglycerides/HDL-cholesterol)]: theoretical and practical implications. Clin Chem 2004; 50: 1113-1115.
    CrossrefPubMedSearch in Google Scholar
  • 30 Csongrádi É, Nagy Jr B, Fülöp T. et al. Increased levels of platelet activation markers are positively associated with carotid wall thickness and other atherosclerotic risk factors in obese patients. Thromb Haemost 2011; 106: 683-692.
    Thieme ConnectPubMedSearch in Google Scholar
  • 31 Stratz C, Nührenberg TG, Binder H. et al. Micro-array profiling exhibits remarkable intra-individual stability of human platelet micro-RNA. Thromb Haemost 2012; 107: 634-641.
    Thieme ConnectPubMedSearch in Google Scholar
  • 32 Zampetaki A, Kiechl S, Drozdov I. et al. Plasma microRNA profiling reveals loss of endothelial miR-126 and other microRNAs in type 2 diabetes. Circ Res 2010; 107: 810-817.
    CrossrefPubMedSearch in Google Scholar
  • 33 Kaudewitz D, Skroblin P, Bender LH. et al. Association of MicroRNAs and YRNAs With Platelet Function. Circ Res 2016; 118: 420-432.
    CrossrefPubMedSearch in Google Scholar
  • 34 Zampetaki A, Willeit P, Tilling L. et al. Prospective study on circulating MicroRNAs and risk of myocardial infarction. J Am Coll Cardiol 2012; 60: 290-299.
    CrossrefPubMedSearch in Google Scholar
  • 35 Edelstein LC, McKenzie SE, Shaw C. et al. MicroRNAs in platelet production and activation. J Thromb Haemost 2013; 11 (Suppl. 01) 340-350.
    CrossrefPubMedSearch in Google Scholar
  • 36 Pescador N, Pérez-Barba M, Ibarra JM. et al. Serum circulating microRNA profiling for identification of potential type 2 diabetes and obesity biomarkers. PLoS One 2013; 08: e77251.
    CrossrefPubMedSearch in Google Scholar
  • 37 Leierseder S, Petzold T, Zhang L. et al. MiR-223 is dispensable for platelet production and function in mice. Thromb Haemost 2013; 110: 1207-1214.
    Thieme ConnectPubMedSearch in Google Scholar
  • 38 Schneider DJ. Factors contributing to increased platelet reactivity in people with diabetes. Diabetes Care 2009; 32: 525-527.
    CrossrefPubMedSearch in Google Scholar
  • 39 Simon LM, Edelstein LC, Nagalla S. et al. Human platelet microRNA-mRNA networks associated with age and gender revealed by integrated plateletomics. Blood 2014; 123: e37-45.
    CrossrefPubMedSearch in Google Scholar
  • 40 Willeit P, Zampetaki A, Dudek K. et al. Circulating microRNAs as novel biomarkers for platelet activation. Circ Res 2013; 112: 595-600.
    CrossrefPubMedSearch in Google Scholar
  • 41 Stratz C, Nührenberg T, Fiebich BL. et al. Controlled type II diabetes mellitus has no major influence on platelet micro-RNA expression. Results from micro-array profiling in a cohort of 60 patients. Thromb Haemost 2014; 111: 902-911.
    Thieme ConnectPubMedSearch in Google Scholar
  • 42 Wang L, Erling P, Bengtsson AA. et al. Transcriptional down-regulation of the platelet ADP receptor P2Y(12) and clusterin in patients with systemic lupus erythematosus. J Thromb Haemost 2004; 02: 1436-1442.
    CrossrefPubMedSearch in Google Scholar
  • 43 Shi R, Zhou X, Ji WJ. et al. The Emerging Role of miR-223 in Platelet Reactivity: Implications in Antiplatelet Therapy. Biomed Res Int 2015; 2015: 981841.
    PubMedSearch in Google Scholar
  • 44 Zhang YY, Zhou X, Ji WJ. et al. Decreased circulating microRNA-223 level predicts high on-treatment platelet reactivity in patients with troponin-negative non-ST elevation acute coronary syndrome. J Thromb Thrombolysis 2014; 38: 65-72.
    CrossrefPubMedSearch in Google Scholar
  • 45 Jones MR, Quinton LJ, Blahna MT. et al. Zcchc11-dependent uridylation of microRNA directs cytokine expression. Nat Cell Biol 2009; 11: 1157-1163.
    CrossrefPubMedSearch in Google Scholar
  • 46 Káplár M, Kappelmayer J, Veszprémi A. et al. The possible association of in vivo leukocyte-platelet heterophilic aggregate formation and the development of diabetic angiopathy. Platelets 2001; 12: 419-422.
    CrossrefPubMedSearch in Google Scholar
  • 47 Ye EA, Steinle JJ. miR-146a Attenuates Inflammatory Pathways Mediated by TLR4/NF-?B and TNFa to Protect Primary Human Retinal Microvascular Endothelial Cells Grown in High Glucose. Mediators Inflamm 2016; 2016: 3958453.
    PubMedSearch in Google Scholar
  • 48 Xiang Y, Cheng J, Wang D. et al. Hyperglycaemia repression of miR-24 coordinately upregulates endothelial cell expression and secretion of von Willebrand factor. Blood 2015; 125: 3377-3387.
    CrossrefPubMedSearch in Google Scholar
  • 49 Finn NA, Eapen D, Manocha P. et al. Coronary heart disease alters intercellular communication by modifying microparticle-mediated microRNA transport. FEBS Lett 2013; 587: 3456-3463.
    CrossrefPubMedSearch in Google Scholar
  • 50 Randriamboavonjy V, Isaak J, Elgheznawy A. et al. Calpain inhibition stabilizes the platelet proteome and reactivity in diabetes. Blood 2012; 120: 415-423.
    CrossrefPubMedSearch in Google Scholar