Involvement of angiotensin II and beta-adrenergic receptors in the regulation of autophagy in human endothelial EA.hy926 cell line

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, CGP42112A, 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.


INTRODUCTION
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]. In autophagy, it is important to decipher the role of autophagosome formation markers, such as microtubule-associated light chain-3 (LC3) and beclin 1, and the autophagy flux marker, p62. Autophagy plays a vital role in maintaining the structural and functional integrity of the endothelial cells [3]. The role of autophagy includes nitric oxide production during starvation [3], vasoreactivity independent of the changes in contractile apparatus, maintenance of metabolic homeostasis [4], regulation of pro-inflammatory and pro-thrombotic functions [5], endothelial cell maturation, senescence, migration and angiogenesis [6].
Alterations in the mechanistic pathway of endothelial cell autophagy is involved in the endothelial dysfunction associated with diabetes mellitus [7] and aging [8]. However, an exploration of the literature review indicates very little information regarding the factors affecting autophagy in the vascular endothelial cells, which may be helpful in preserving the endothelial functions and reducing the cardiovascular risks.
It is reported that angiotensin II (Ang II) induces autophagy in the rat cardiomyocytes [9], vascular smooth muscle cells [10], and podocytes [11]. Porrello et al demonstrated that the stimulation of the Ang II type 1 receptor (AT1R) increases autophagy, whereas Ang II type 2 receptor (AT2R) subdues it in neonatal cardiomyocytes [12]. The AT1R also mediates mechanical stress induced autophagy in the cardiomyocytes [13]. Isoproterenol-induced βAR stimulation is also known to trigger autophagy in cardiac fibroblast cells [14]. The stimulation of type 2 βAR (β2AR) provokes autophagy in the hepatic cells [15] and cardiac fibroblasts [14]. 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]. However, despite extensive clinical experience with pharmacological agents that act through the Ang II receptors or βAR and their effects on the endothelial cell autophagy has not been studied. Thus, 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.

In vitro cell culture
The human endothelial EA.hy926 cell line was provided as a gift from Dr. C. J. Edgell, the University of North Carolina, Lineberger Comprehensive Cancer Centre, Chapel Hill, United States [17]. The cells were cultured in complete Dulbecco's Modified Eagle Medium (DMEM, Himedia, India) supplemented with 10 % fetal bovine serum (FBS, Himedia, India) and 1 % Penicillin/Streptomycin (Himedia, India). It was incubated at 37 °C in 5 % CO2 until they reached 80 -90 % confluence. The cultured cells were then randomly exposed to following treatments: Ang II, candesartan, PD1232319, CGP-42112A, isoproterenol, propranolol, dobutamine, metoprolol, salbutamol and ICI-118,551 as described below under respective assay.

Cell viability assay
Cytotoxicity of the human endothelial EA.hy926 cells in response to the pharmacological modulators was determined by 3-[4,5dimethylthiazol-2-y]-2, 5-diphenyltetrazolium bromide (MTT) assay. The principle behind the assay is directly dependent on the metabolic reduction of MTT. A total of 2 x 10 4 cells were seeded in 96 well plates and were left overnight to adhere. The cells were then exposed for 24 h to following treatments; 1 nM to 10 µM: Ang II After the treatment, the cells were then incubated with MTT for 2-3 h (0.5 mg/L) followed by the addition of 150 μL of dimethylsulphoxide to each well. A microplate reader (BioTek, USA) was used to record the changes in absorbance at 570 nm. The data is presented as the percent cell viability normalized to their respective controls.

Immunoblotting
The cell lysates containing 40 µg of the total proteins were fractionated on a 12 % SDS-PAGE.
The fractionated proteins were transferred to a polyvinylidene fluoride membrane. The blots were then blocked at room temperature with a solution consisting of 5 % bovine serum albumin in Tris-buffered saline (TBS) containing 0.1 % Tween 20 for 2 h. Following which the membranes were incubated overnight with the diluted antibodies: anti-LC3B (1:1000 dilution; Cell Signalling Technology, USA), anti-p62 (1:1000 dilution; Sigma, USA) and anti-beta actin (1:10000 dilution; Sigma, USA).
The membranes were then incubated for 2 h with the respective HRP-conjugated mouse or rabbit secondary antibodies and visualized using ECL western-blotting detection reagent. The intensity of the immunoblots was analysed with myImageAnalysis software version 2.0 (Thermo Scientific, USA).

Statistical analysis
The results are expressed as mean ± standard error of the mean (S.E.M). In order to investigate the variability amongst the groups, one-way ANOVA was employed. Bonferroni's test was used for the post hoc analysis when the F-test of variance had achieved the necessary level of statistical significance (i.e., p < 0.05). The statistical testing was done using the Graph Pad prism software version 5.00.28 (GraphPad Software, Inc, San Diego, CA, USA). A level of the probability less than 0.05 was considered significant.

Angiotensin II upregulated endothelial cell autophagy via the AT1R stimulation
Angiotensin II (1 nM -10 µM) exposure for 24 h did not affect the viability of the human endothelial EA.hy.926 cell line (Figure 1 A). Candesartan showed no sign of toxicity at a concentration of 1nM to 1 µM, but its 10 µM showed a significant reduction in cell viability (Figure 1 B). Angiotensin II at 1 µM concentration significantly increased LC3B-II and decreased p62 (Figure 1 C). It was observed that cotreatment with the AT1R blocker, candesartan (1 µM) significantly inhibited the Ang II (1 µM)enhanced expression of the LC3B-II (Figure 1 D). Reduction in p62 expression by Ang II was also significantly suppressed on cotreatment with candesartan at 100 nM and 1 µM.

Activation of β1AR suppressed autophagy in endothelial cells
No significant cytotoxic effect on human endothelial EA.hy926 cells was observed upon 24 h treatment with selective β1AR agonist dobutamine (Figures 4 A) and selective β1AR antagonist metoprolol (Figures 4 B)

DISCUSSION
A detailed understanding of the molecular aspect of autophagy pathway is evolving, but limited information is available about the cardiovascular cues influencing autophagy in the endothelial cells. The present study is the first to report about the involvement of the Ang II receptors and βARs in the regulation of endothelial cell autophagy. ; * p < 0.05, ** p < 0.01 and *** p < 0.005 when compared to control and # p < 0.05, ## p < 0.01 and ### p < 0.005 when compared to salbutamol-treated cells (n = 5) Angiotensin II was found to induce autophagy in the endothelial cells via AT1R stimulation, while AT2R activation antagonized it. The activation of β1AR suppressed the endothelial cell autophagy; whereas, β2AR activation induced it. Angiotensin II, the vasoactive peptide of RAAS implicated in various vascular pathologies, including hypertension, has been documented to trigger autophagy in tissues of cardiovascular origin, namely cardiomyocytes [12], cardiac fibroblasts [18], vascular smooth muscle cells [19], and endothelial cells [20]. In line with the earlier studies, Ang II-treated endothelial cells showed increase in autophagosome formation and the autophagy flux as indicated by the increased LC3B-II and reduced p62 expression. There was observed suppression of Ang II-induced endothelial cell autophagy upon treatment with candesartan, a selective AT1R antagonist. This indicates that the AT1R activation mediates the pro-autophagic effect of Ang II on the endothelial cells.
Treatment with PD123319, a selective AT2R blocker, resisted the inhibitory effect of candesartan on the Ang II-induced autophagy. This suggests that AT2Rs may also be involved in regulating the effect of Ang II on endothelial cell autophagy. The inhibitory effect of AT2R stimulation on the Ang II-induced endothelial cell autophagy was confirmed when cotreatment with CGP-4211A, a selective AT2R activator, suppressed the Ang II-induced endothelial cell autophagy. 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 AT2R overexpression in cardiac cells counteracts the effect of Ang II [12].
Sympathetic hyperactivity is another significant stressor affecting vascular health during cardiovascular diseases, the landmark of which is tachycardia due to the sustained βAR activation. In a recent study, isoproterenol was found to induce vascular oxidative stress and endothelial dysfunction via β2AR [21]. Given that the autophagy is a cellular stress response mechanism, which is also involved in regulating endothelial morphology and functions, the understanding of the effect of the βAR stimulation on autophagy in the endothelial cells becomes important.
In the present study, the isoproterenol treatment increased LC3B-II and reduced p62 expression in the endothelial cells, indicating upregulation of autophagy in the cells. Cotreatment with propranolol, a non-selective βAR, significantly suppressed the autophagy inducing effect of isoproterenol indicating that the βARs could be involved in modulating the endothelial cell autophagy. The role of β1ARs and β2ARs in modulating endothelial cell autophagy was investigated by treating the cells with dobutamine (selective β1AR agonist) in absence or presence of metoprolol (selective β1AR antagonist) and salbutamol (selective β2AR agonist) in absence or presence of ICI-118,551 (selective β2AR antagonist), respectively [22].
Dobutamine treatment reduced the LC3B-II expression and increased p62 expression in the endothelial cells, which marks the inhibition of autophagy. The inhibitory effect of dobutamine on endothelial cell autophagy was suppressed upon cotreatment with metoprolol, which indicates β1AR activation exhibits anti-autophagic effect in the endothelial cells. This goes in line with the findings of Wang and his colleagues in 2013 and 2018, in which they revealed that β1AR activation by anti β1AR autoantibodies in the cardiomyocytes inhibits autophagy [16,23]. Treatment with salbutamol enhanced the expression of LC3B-II expression and reduced p62 expression in the endothelial cells autophagy, indicating induction of autophagy. The autophagy stimulatory effect of salbutamol was counteracted upon the selective β2AR blockade by ICI-118,551, which suggests proautophagic effect of β2AR activation in the endothelial cells. Similar findings were observed in a report published by Farah and colleagues in 2014, demonstrating the induction of autophagy by activation of β2ARs in the hepatic cells [15].
Earlier reports also demonstrated that the majority of the βAR population on the vascular endothelium is of the β2AR subtype, which may also be the reason for the predominance of the pro-autophagic effect upon βAR activation by isoproterenol in the endothelial cells [24]. Aranguiz-Urroz and colleagues (2015) observed upregulation of autophagy in the cardiac fibroblasts upon exposure to isoproterenol, which was blocked by the selective β2AR antagonist ICI-118,551, but not by atenolol (selective β1AR blocker). Thereby suggesting that β2AR activation mediates the autophagy stimulatory effect of isoproterenol in the cardiac fibroblasts [14]. From the above findings, it could be summarized that β1AR stimulation results in antiautophagic effect, whereas β2AR activation has pro-autophagic effect in the endothelial cells.

CONCLUSION
Autophagy plays a crucial role in regulating the endothelial cell biology, damage, dysfunction, survival, and apoptosis, and thus, could be an important contributor in the development of cardiovascular diseases. However, knowledge about the proximal cues affecting autophagy in the endothelial cells is limited. The present study revealed that AT1R and AT2R act reciprocally to regulate Ang II mediated autophagy in the endothelial cells. AT1R mediates Ang II-induced autophagy, while AT2R antagonizes it. It was also observed that β1AR stimulation suppresses the endothelial cell autophagy, while β2AR activation triggers it. These novel findings aid in understanding the roles of Ang II receptors as well as βARs in regulating autophagy in the endothelial cells that could be exploited therapeutically to lower cardiovascular disease risks.