Abstract
Purpose: To investigate the role of angiotensin II (Ang II) and β adrenergic receptors (βARs) in autophagy regulation in human endothelial EA.hy926 cell line.Methods: The effect of pharmacological modulation of Ang II receptors and βARs on the expression of LC3B-II and p62 proteins (autophagosome formation marker and autophagic flux marker, respectively) in the human endothelial EA.hy926 cell line were investigated by immunoblotting technique.Results: Ang II-induced autophagy was characterized by increased LC3B-II and reduced p62 expressions. Candesartan, an AT1R agonist, significantly suppressed the effects of Ang II, while a selective AT2R antagonist, PD123319, inhibited the effect of candesartan. An AT2R agonist, CGP-42112A, also suppressed the Ang II-induced autophagy. Treatment with isoproterenol enhanced the expression of LC3B-II and reduced that of p62; these effects were suppressed upon cotreatment with propranolol (non-selective βAR blocker propranolol). A selective β1AR agonist, dobutamine, reduced the expression of LC3B-II, and increased that of p62; the same was suppressed upon treatment with a selective β1AR antagonist, metoprolol. A selective β2AR agonist, salbutamol, resulted in increased expression of LC3B-II and reduced expression of p62. These effects were encountered upon treatment with selective β2AR antagonist, ICI-118,551.Conclusion: Based on the foregoing, it is evident that AT1Rs mediates Ang II-induced endothelial cell autophagy, while AT2Rs antagonizes the mechanism. βAR activation mediates isoproterenol-induced endothelial cell autophagy, which results from the balance of β1ARs-mediated suppression and β2ARsmediated upregulation of autophagy in the endothelial cells.
 Keywords: Autophagy, Angiotensin II type 1 receptors, Angiotensin II type 2 receptors β adrenergic type 1 receptors, β adrenergic type 2 receptors endothelial cells
Highlights
Autophagy is a regulated intracellular process in which damaged organelles and misfolded proteins are recycled in a lysosome-dependent manner [1]
It was observed that cotreatment with the Ang II type 1 receptor (AT1R) blocker, candesartan (1 μM) significantly inhibited the angiotensin II (Ang II) (1 μM)enhanced expression of the LC3B-II (Figure 1 D)
This finding goes in line with the previous study of Porello et al reporting that the AT1R stimulation by Ang II increases the autophagosome formation in the neonatal rat cardiomyocytes, whereas Ang II type 2 receptor (AT2R) overexpression in cardiac cells counteracts the effect of Ang II [12]
Summary
Autophagy is a regulated intracellular process in which damaged organelles and misfolded proteins are recycled in a lysosome-dependent manner [1]. It is an essential mechanism for the maintenance of cellular homeostasis and stress response [2]. Type 1 βAR (β1AR) stimulation by anti β1AR autoantibodies causes a reduction in autophagy in the cardiomyocytes resulting into cell death followed by the reduced cardiac function [16]. The present study aims to investigate the role of subtypes of Ang II receptor and βAR in mechanistic regulation of autophagy in the human endothelial EA.hy926 cell line using their pharmacological modulators
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