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Purinergic Signalling

, Volume 14, Issue 2, pp 141–156 | Cite as

Epac is required for exogenous and endogenous stimulation of adenosine A2B receptor for inhibition of angiotensin II-induced collagen synthesis and myofibroblast differentiation

  • Sarawuth Phosri
  • Kwanchai Bunrukchai
  • Warisara Parichatikanond
  • Vilasinee H. Sato
  • Supachoke Mangmool
Original Article
  • 202 Downloads

Abstract

Angiotensin II (Ang II) plays an important role on the pathogenesis of cardiac fibrosis. Prolong and overstimulation of angiotensin II type 1 receptor with Ang II-induced collagen synthesis and myofibroblast differentiation in cardiac fibroblasts, leading to cardiac fibrosis. Although adenosine and its analogues are known to have cardioprotective effects, the mechanistic by which adenosine A2 receptors (A2Rs) inhibit Ang II-induced cardiac fibrosis is not clearly understood. In the present study, we examined the effects of exogenous adenosine and endogenous adenosine on Ang II-induced collagen and myofibroblast differentiation determined by α-smooth muscle action (α-SMA) overexpression and their underlying signal transduction. Elevation of endogenous adenosine levels resulted in the inhibition of Ang II-induced collagen type I and III and α-SMA synthesis in cardiac fibroblasts. Moreover, treatment with exogenous adenosine which selectively stimulated A2Rs also suppressed Ang II-induced collagen synthesis and α-SMA production. These antifibrotic effects of both endogenous and exogenous adenosines are mediated through the A2B receptor (A2BR) subtype. Stimulation of A2BR exhibited antifibrotic effects via the cAMP-dependent and Epac-dependent pathways. Our results provide new mechanistic insights regarding the role for cAMP and Epac on A2BR-mediated antifibrotic effects. Thus, A2BR is one of the potential therapeutic targets against cardiac fibrosis.

Keywords

Adenosine A2B receptor Angiotensin II Cardiac fibrosis Epac cAMP Collagen α-SMA 

Abbreviations

α-SMA

α-Smooth muscle actin

AC

Adenylate cyclase

A2R

Adenosine A2 receptor

Ang II

Angiotensin II

cAMP

Cyclic adenosine monophosphate

DDA

2′,5′-Dideoxyadenosine

ECM

Extracellular matrix

EHNA

Erythro-9-(2-hydroxy-3-nonyl) adenine

Epac

Exchange protein directly activated by cAMP

ET-1

Endothelin-1

FCS

Fetal calf serum

IDO

5-Iodotubericidin

PKA

Protein kinase A

PKI

Myristoylated protein kinase A inhibitor amide 14–22

siRNA

Small interfering RNA

TGF-β

Transforming growth factor beta

Notes

Acknowledgements

This study is funded by the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program, the Office of the Higher Education Commission, Ministry of Education [Grant PHD/0179/2558] (to S.P.) and TRF Grant [Grant RSA6080061] (to S.M.). This study is part of a Ph.D. thesis of Mahidol University.

Author contributions

Participated in the study planning: S.P. and S.M.

Performed experiments and data analysis: S.P., K.B., and S.M.

Contributed to the discussion and reviewed/edited the manuscript: W.P. and V.H.S.

Wrote the manuscript: S.P. and S.M.

Compliance with ethical standards

Conflicts of interest

Sarawuth Phosri declares that he has no conflict of interest.

Kwanchai Bunrukchai declares that he has no conflict of interest.

Warisara Parichatikanond declares that she has no conflict of interest.

Vilasinee H. Sato declares that she has no conflict of interest.

Supachoke Mangmool declares that he has no conflict of interest.

Ethical approval

This study was approved by the Institutional Animal Care and Use Committee of the Faculty of Pharmacy, Mahidol University (Protocol Nos. PYR005/2557 and PYR002/2560).

Supplementary material

11302_2017_9600_MOESM1_ESM.docx (16 kb)
ESM 1 (DOCX 16 kb)
11302_2017_9600_FIG9_ESM.gif (46 kb)
Fig. S1

Effects of endogenous adenosine on Ang II-induced collagen and α-SMA synthesis. Cardiac fibroblasts were either pretreated with EHNA (1, 10 μM) or IDO (1, 10 μM) for 1 h and then stimulated with 200 nM Ang II for 6 h. Relative collagen I, collagen III, and α-SMA mRNA levels were quantified and shown as the mean±SEM (n = 4). * Significant difference (P < 0.05) versus vehicle (control); #Significant difference (P < 0.05) versus Ang II. (GIF 45 kb)

11302_2017_9600_MOESM2_ESM.tif (301 kb)
High resolution (TIFF 301 kb)

References

  1. 1.
    Porter KE, Turner NA (2009) Cardiac fibroblasts: at the heart of myocardial remodeling. Pharmacol Ther 123(2):255–278.  https://doi.org/10.1016/j.pharmthera.2009.05.002 CrossRefPubMedGoogle Scholar
  2. 2.
    Vasquez C, Benamer N, Morley GE (2011) The cardiac fibroblast: functional and electrophysiological considerations in healthy and diseased hearts. J Cardiovasc Pharmacol 57(4):380–388.  https://doi.org/10.1097/FJC.0b013e31820cda19 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Kong P, Christia P, Frangogiannis NG (2014) The pathogenesis of cardiac fibrosis. Cell Mol Life Sci 71(4):549–574.  https://doi.org/10.1007/s00018-013-1349-6 CrossRefPubMedGoogle Scholar
  4. 4.
    Kurose H, Mangmool S (2016) Myofibroblasts and inflammatory cells as players of cardiac fibrosis. Arch Pharm Res 39(8):1100–1113.  https://doi.org/10.1007/s12272-016-0809-6 CrossRefPubMedGoogle Scholar
  5. 5.
    Lijnen PJ, Petrov VV, Fagard RH (2000) Induction of cardiac fibrosis by angiotensin II. Methods Find Exp Clin Pharmacol 22(10):709–723.  https://doi.org/10.1358/mf.2000.22.10.802287 CrossRefPubMedGoogle Scholar
  6. 6.
    Holtz J (1993) Pathophysiology of heart failure and the renin-angiotensin-system. Basic Res Cardiol 88(Suppl 1):183–201PubMedGoogle Scholar
  7. 7.
    Pedram A, Razandi M, O'Mahony F, Lubahn D, Levin ER (2010) Estrogen receptor-beta prevents cardiac fibrosis. Mol Endocrinol 24(11):2152–2165.  https://doi.org/10.1210/me.2010-0154 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Schnee JM, Hsueh WA (2000) Angiotensin II, adhesion, and cardiac fibrosis. Cardiovasc Res 46(2):264–268.  https://doi.org/10.1016/S0008-6363(00)00044-4 CrossRefPubMedGoogle Scholar
  9. 9.
    Crawford DC, Chobanian AV, Brecher P (1994) Angiotensin II induces fibronectin expression associated with cardiac fibrosis in the rat. Circ Res 74(4):727–739.  https://doi.org/10.1161/01.RES.74.4.727 CrossRefPubMedGoogle Scholar
  10. 10.
    Kawano H, Cody RJ, Graf K, Goetze S, Kawano Y, Schnee J, Law RE, Hsueh WA (2000) Angiotensin II enhances integrin and alpha-actinin expression in adult rat cardiac fibroblasts. Hypertension 35(1):273–279.  https://doi.org/10.1161/01.HYP.35.1.273 CrossRefPubMedGoogle Scholar
  11. 11.
    Villarreal F, Zimmermann S, Makhsudova L, Montag AC, Erion MD, Bullough DA, Ito BR (2003) Modulation of cardiac remodeling by adenosine: in vitro and in vivo effects. Mol Cell Biochem 251(1/2):17–26.  https://doi.org/10.1023/A:1025401026441 CrossRefPubMedGoogle Scholar
  12. 12.
    Phosri S, Arieyawong A, Bunrukchai K, Parichatikanond W, Nishimura A, Nishida M, Mangmool S (2017) Stimulation of adenosine A2B receptor inhibits Endothelin-1-induced cardiac fibroblast proliferation and alpha-smooth muscle actin synthesis through the cAMP/Epac/PI3K/Akt-signaling pathway. Front Pharmacol 8:428.  https://doi.org/10.3389/fphar.2017.00428 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Dubey RK, Gillespie DG, Zacharia LC, Mi Z, Jackson EK (2001) A(2b) receptors mediate the antimitogenic effects of adenosine in cardiac fibroblasts. Hypertension 37(2):716–721.  https://doi.org/10.1161/01.HYP.37.2.716 CrossRefPubMedGoogle Scholar
  14. 14.
    Villarreal F, Epperson SA, Ramirez-Sanchez I, Yamazaki KG, Brunton LL (2009) Regulation of cardiac fibroblast collagen synthesis by adenosine: roles for Epac and PI3K. Am J Phys Cell Physiol 296(5):C1178–C1184.  https://doi.org/10.1152/ajpcell.00291.2008 CrossRefGoogle Scholar
  15. 15.
    Epperson SA, Brunton LL, Ramirez-Sanchez I, Villarreal F (2009) Adenosine receptors and second messenger signaling pathways in rat cardiac fibroblasts. Am J Phys Cell Physiol 296(5):C1171–C1177.  https://doi.org/10.1152/ajpcell.00290.2008 CrossRefGoogle Scholar
  16. 16.
    Dubey RK, Gillespie DG, Jackson EK (1998) Adenosine inhibits collagen and protein synthesis in cardiac fibroblasts: role of A2B receptors. Hypertension 31(4):943–948.  https://doi.org/10.1161/01.HYP.31.4.943 CrossRefPubMedGoogle Scholar
  17. 17.
    Dubey RK, Gillespie DG, Mi Z, Jackson EK (1997) Exogenous and endogenous adenosine inhibits fetal calf serum-induced growth of rat cardiac fibroblasts: role of A2B receptors. Circulation 96(8):2656–2666.  https://doi.org/10.1161/01.CIR.96.8.2656 CrossRefPubMedGoogle Scholar
  18. 18.
    Smolenski RT, Kochan Z, McDouall R, Page C, Seymour AL, Yacoub MH (1994) Endothelial nucleotide catabolism and adenosine production. Cardiovasc Res 28(1):100–104.  https://doi.org/10.1093/cvr/28.1.100 CrossRefPubMedGoogle Scholar
  19. 19.
    Cohen MV, Downey JM (2008) Adenosine: trigger and mediator of cardioprotection. Basic Res Cardiol 103(3):203–215.  https://doi.org/10.1007/s00395-007-0687-7 CrossRefPubMedGoogle Scholar
  20. 20.
    Jacobson KA, Gao ZG (2006) Adenosine receptors as therapeutic targets. Nat Rev Drug Discov 5(3):247–264.  https://doi.org/10.1038/nrd1983 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Vecchio EA, Chuo CH, Baltos JA, Ford L, Scammells PJ, Wang BH, Christopoulos A, White PJ, May LT (2016) The hybrid molecule, VCP746, is a potent adenosine A2B receptor agonist that stimulates anti-fibrotic signalling. Biochem Pharmacol 117:46–56.  https://doi.org/10.1016/j.bcp.2016.08.007 CrossRefPubMedGoogle Scholar
  22. 22.
    Wakeno M, Minamino T, Seguchi O, Okazaki H, Tsukamoto O, Okada K, Hirata A, Fujita M, Asanuma H, Kim J, Komamura K, Takashima S, Mochizuki N, Kitakaze M (2006) Long-term stimulation of adenosine A2b receptors begun after myocardial infarction prevents cardiac remodeling in rats. Circulation 114(18):1923–1932.  https://doi.org/10.1161/CIRCULATIONAHA.106.630087 CrossRefPubMedGoogle Scholar
  23. 23.
    Klinger M, Freissmuth M, Nanoff C (2002) Adenosine receptors: G protein-mediated signalling and the role of accessory proteins. Cell Signal 14(2):99–108.  https://doi.org/10.1016/S0898-6568(01)00235-2 CrossRefPubMedGoogle Scholar
  24. 24.
    Swaney JS, Roth DM, Olson ER, Naugle JE, Meszaros JG, Insel PA (2005) Inhibition of cardiac myofibroblast formation and collagen synthesis by activation and overexpression of adenylyl cyclase. Proc Natl Acad Sci U S A 102(2):437–442.  https://doi.org/10.1073/pnas.0408704102 CrossRefPubMedGoogle Scholar
  25. 25.
    Lu D, Aroonsakool N, Yokoyama U, Patel HH, Insel PA (2013) Increase in cellular cyclic AMP concentrations reverses the profibrogenic phenotype of cardiac myofibroblasts: a novel therapeutic approach for cardiac fibrosis. Mol Pharmacol 84(6):787–793.  https://doi.org/10.1124/mol.113.087742 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Nuamnaichati N, Sato VH, Moongkarndi P, Parichatikanond W, Mangmool S (2017) Sustained β-AR stimulation induces synthesis and secretion of growth factors in cardiac myocytes that affect on cardiac fibroblast activation. Life Sci.  https://doi.org/10.1016/j.lfs.2017.10.034
  27. 27.
    Mangmool S, Denkaew T, Phosri S, Pinthong D, Parichatikanond W, Shimauchi T, Nishida M (2016) Sustained βAR stimulation mediates cardiac insulin resistance in a PKA-dependent manner. Mol Endocrinol 30(1):118–132.  https://doi.org/10.1210/me.2015-1201 CrossRefPubMedGoogle Scholar
  28. 28.
    Insel PA, Murray F, Yokoyama U, Romano S, Yun H, Brown L, Snead A, Lu D, Aroonsakool N (2012) cAMP and Epac in the regulation of tissue fibrosis. Br J Pharmacol 166(2):447–456.  https://doi.org/10.1111/j.1476-5381.2012.01847.x CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Kawano H, Do YS, Kawano Y, Starnes V, Barr M, Law RE, Hsueh WA (2000) Angiotensin II has multiple profibrotic effects in human cardiac fibroblasts. Circulation 101(10):1130–1137.  https://doi.org/10.1161/01.CIR.101.10.1130 CrossRefPubMedGoogle Scholar
  30. 30.
    Schieffer B, Wirger A, Meybrunn M, Seitz S, Holtz J, Riede UN, Drexler H (1994) Comparative effects of chronic angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade on cardiac remodeling after myocardial infarction in the rat. Circulation 89(5):2273–2282.  https://doi.org/10.1161/01.CIR.89.5.2273 CrossRefPubMedGoogle Scholar
  31. 31.
    Regan CP, Anderson PG, Bishop SP, Berecek KH (1997) Pressure-independent effects of AT1-receptor antagonism on cardiovascular remodeling in aortic-banded rats. Am J Phys 272:H2131–H2138Google Scholar
  32. 32.
    Fredholm BB, Arslan G, Halldner L, Kull B, Schulte G, Wasserman W (2000) Structure and function of adenosine receptors and their genes. Naunyn Schmiedeberg's Arch Pharmacol 362(4-5):364–374.  https://doi.org/10.1007/s002100000313 CrossRefGoogle Scholar
  33. 33.
    Vecchio EA, White PJ, May LT (2017) Targeting adenosine receptors for the treatment of cardiac fibrosis. Front Pharmacol 8:243.  https://doi.org/10.3389/fphar.2017.00243 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Zimmerman MA, Tak E, Ehrentraut SF, Kaplan M, Giebler A, Weng T, Choi DS, Blackburn MR, Kam I, Eltzschig HK, Grenz A (2013) Equilibrative nucleoside transporter (ENT)-1-dependent elevation of extracellular adenosine protects the liver during ischemia and reperfusion. Hepatology 58(5):1766–1778.  https://doi.org/10.1002/hep.26505 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Eltzschig HK, Faigle M, Knapp S, Karhausen J, Ibla J, Rosenberger P, Odegard KC, Laussen PC, Thompson LF, Colgan SP (2006) Endothelial catabolism of extracellular adenosine during hypoxia: the role of surface adenosine deaminase and CD26. Blood 108(5):1602–1610.  https://doi.org/10.1182/blood-2006-02-001016 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Jackson EK, Koehler M, Mi Z, Dubey RK, Tofovic SP, Carcillo JA, Jones GS (1996) Possible role of adenosine deaminase in vaso-occlusive diseases. J Hypertens 14(1):19–29CrossRefPubMedGoogle Scholar
  37. 37.
    Hori M, Kitakaze M (1991) Adenosine, the heart, and coronary circulation. Hypertension 18(5):565–574.  https://doi.org/10.1161/01.HYP.18.5.565 CrossRefPubMedGoogle Scholar
  38. 38.
    Lagerkranser M, Sollevi A, Irestedt L, Tidgren B, Andreen M (1985) Renin release during controlled hypotension with sodium nitroprusside, nitroglycerin and adenosine: a comparative study in the dog. Act Aneasthesiol Scand 29(1):45–49.  https://doi.org/10.1111/j.1399-6576.1985.tb02157.x CrossRefGoogle Scholar
  39. 39.
    Chen Y, Epperson S, Makhsudova L, Ito B, Suarez J, Dillmann W, Villarreal F (2004) Functional effects of enhancing or silencing adenosine A2b receptors in cardiac fibroblasts. Am J Physiol Heart Circ Physiol 287(6):H2478–H2486.  https://doi.org/10.1152/ajpheart.00217.2004 CrossRefPubMedGoogle Scholar
  40. 40.
    Asakura M, Asanuma H, Kim J, Liao Y, Nakamaru K, Fujita M, Komamura K, Isomura T, Furukawa H, Tomoike H, Hitakaze M (2007) Impact of adenosine receptor signaling and metabolism on pathophysiology in patients with chronic heart failure. Hypertens Res 30(9):781–787.  https://doi.org/10.1291/hypres.30.781 CrossRefPubMedGoogle Scholar
  41. 41.
    Shaikh G, Cronstein B (2016) Signaling pathways involving adenosine A2A and A2B receptors in wound healing and fibrosis. Purinergic Signal 12(2):191–197.  https://doi.org/10.1007/s11302-016-9498-3 CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Godinho RO, Duarte T, Pacini ES (2015) New perspectives in signaling mediated by receptors coupled to stimulatory G protein: the emerging significance of cAMP e ffl ux and extracellular cAMP-adenosine pathway. Front Pharmacol 6:58CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Fang Y, Olah ME (2007) Cyclic AMP-dependent, protein kinase A-independent activation of extracellular signal-regulated kinase 1/2 following adenosine receptor stimulation in human umbilical vein endothelial cells: role of exchange protein activated by cAMP 1 (Epac1). J Pharmacol Exp Ther 322(3):1189–1200.  https://doi.org/10.1124/jpet.107.119933 CrossRefPubMedGoogle Scholar

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Authors and Affiliations

  1. 1.Department of Pharmacology, Faculty of PharmacyMahidol UniversityBangkokThailand

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