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. 2019 Sep;15(3):357-366.
doi: 10.1007/s11302-019-09668-z. Epub 2019 Jun 28.

Multiple adenosine receptor subtypes stimulate wound healing in human EA.hy926 endothelial cells

Zeinab Bonyanian  1 , Matthew Walker  1 , Eugene Du Toit  1 , Roselyn B Rose'Meyer  2
Affiliations

Multiple adenosine receptor subtypes stimulate wound healing in human EA.hy926 endothelial cells

Zeinab Bonyanian et al. Purinergic Signal. 2019 Sep.

Abstract

Wound healing is an important outcome of tissue damage and can be stimulated by adenosine released from cells during events such as tissue injury, ischaemia or tumour growth. The aim of this research was to determine the potency and efficacy of adenosine A1, A2A and A2B receptor agonists on the rate of wound healing and cell proliferation in human EA.hy926 endothelial cells. Real-time PCR data showed that only adenosine A1, A2A and A2B receptor mRNA were expressed in this cell line. All three adenosine receptor agonists, CPA, CGS21680 and NECA, significantly increased the rate of wound healing in human EAhy926 endothelial cells with the following order of potency CGS21680>CPA>NECA and efficacy CPA>NECA>CGS21680. The selective adenosine A1, A2A and A2B receptor antagonists, DPCPX, ZM241385 and MRS1754 (all at 10 nM), reversed the effects of their respective agonists. EAhy926 endothelial cell proliferation was also significantly increased with the adenosine A1 and A2B receptor agonists, CPA and NECA. Western blot analysis demonstrated that adenosine A2A and A1 receptor protein levels were highly expressed compared with the adenosine A2B receptors in the EAhy926 endothelial cell lines. While all three adenosine A1, A2A and A2B receptor subtypes contribute to cell proliferation and wound healing in human EAhy926 endothelial cells, treatments selectively targeting receptor subtypes may further enhance wound healing.

Keywords: Adenosine; Cell proliferation; Endothelial cells; Wound healing.

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Conflict of interest statement

Zeinab Bonyanian declares that he has no conflict of interest.

Matthew Walker declares that he has no conflict of interest.

Eugene Du Toit declares that he has no conflict of interest.

Roselyn B. Rose’Meyer declares that she has no conflict of interest.

Figures

Fig. 1
Fig. 1
Upper panel: 2% Agarose gel electrophoresis RT-PCR results for adenosine receptors. 18 s ribosomal RNA 71BP, adenosine A1 receptor 97BP, adenosine A2A receptor 122BP, adenosine A2B receptor 255BP, adenosine A3 receptor 171BP and β(B)-actin 122BP. Lower panel: Graph demonstrating the relative expression of adenosine A1, A2A and A2B receptor subtypes on E.Ahy926 endothelial cells. The data was analysed using one-way ANOVA. Data is expressed as mean ± SEM (n = 6), *P < 0.05 versus adenosine A1 receptor
Fig. 2
Fig. 2
Wound healing scratch assay using different concentrations of CPA on EA.hy926 endothelial cells. Upper panel depicts scratch widths at 0 times and lower panel shows scratch widths after 6 h. CPA concentrations from the left to right are control (0 nM), 1 nM, 3 nM, 10 nM, 30 nM, 100 nM and 300 nM CPA. Magnification, × 40
Fig. 3
Fig. 3
Concentration-response curves (CRC) showing the effect of selective adenosine receptor agonists on EA.hy926 endothelial cells and the rate of wound healing, n = 6 per group. Note that NECA concentration-response curves were done in the presence of 10 nM ZM241385
Fig. 4
Fig. 4
The effect of adenosine receptor agonist, antagonist and their combination on the rate of wound healing. Note that responses to NECA were done in the presence of 10 nM ZM241385. One-way ANOVA was applied to analyse the data. Data is expressed as mean ± SEM, n = 6, *P < 0.05 versus the control, +P < 0.05 versus selective agonist. CGS21680 (CGS), ZM241385 (ZM) and MRS1754 (MRS)
Fig. 5
Fig. 5
The effect of adenosine receptor agonist, antagonist and their combination on proliferation rates. Note that responses to NECA were done in the presence of 10 nM ZM241385. One-way ANOVA was applied to analyse the data. Data is expressed as mean ± SEM, n = 6, *P < 0.05 versus the control. CGS21680 (CGS), ZM241385 (ZM) and MRS1754 (MRS)
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