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

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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.

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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

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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.

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Authors and Affiliations

  1. Department of Pharmacology, Faculty of Pharmacy, Mahidol University, Bangkok, 10400, Thailand

    Sarawuth Phosri, Kwanchai Bunrukchai, Warisara Parichatikanond, Vilasinee H. Sato & Supachoke Mangmool

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  1. Sarawuth Phosri
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  2. Kwanchai Bunrukchai
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  3. Warisara Parichatikanond
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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.

Corresponding author

Correspondence to Supachoke Mangmool.

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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).

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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)

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Phosri, S., Bunrukchai, K., Parichatikanond, W. et al. Epac is required for exogenous and endogenous stimulation of adenosine A2B receptor for inhibition of angiotensin II-induced collagen synthesis and myofibroblast differentiation. Purinergic Signalling 14, 141–156 (2018). https://doi.org/10.1007/s11302-017-9600-5

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