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, 170, 20-28

Effect of Brimonidine, an α2 Adrenergic Agonist, on Human Meibomian Gland Epithelial Cells

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Effect of Brimonidine, an α2 Adrenergic Agonist, on Human Meibomian Gland Epithelial Cells

Xi Han et al. Exp Eye Res.

Abstract

We recently discovered that the anti-glaucoma pharmaceuticals timolol, a β adrenergic antagonist, and pilocarpine, a cholinergic compound, negatively influence the morphology, proliferative capacity and survival of human meibomian gland epithelial cells (HMGECs). We hypothesize that another class of anti-glaucoma drugs, the α2 adrenergic agonists, also acts directly on HMGECs to affect their structure and function. We tested this hypothesis. Immortalized (i) HMGECs were cultured with brimonidine, as well as clonidine (α2 agonist), phenylephrine (α1 agonist), RX821002 (inverse α2 agonist) and MK912 (neutral α2 agonist) for up to 7 days. Cells were counted with a hemocytometer, and evaluated for morphology, signaling pathway activity, protein biomarker expression, and the accumulation of neutral lipids, phospholipids and lysosomes. Our findings demonstrate that brimondine treatment induces a dose-dependent decrease in Akt signaling and proliferation of iHMGECs. In contrast, brimonidine also promotes a dose-dependent differentiation of iHMGECs, including an increase in neutral lipid, phospholipid and lysosome levels. These effects were paralleled by an inhibition of p38 signaling, and duplicated by cellular exposure to clonidine, but not phenylephrine. Brimonidine also enhanced the cellular content of sterol regulatory binding protein-1, a master regulator of lipid synthesis. Of particular interest, the putative α2 antagonists, RX821002 and MK912, did not interfere with brimonidine action, but rather stimulated IHMGEC differentiation. Our results support our hypothesis and demonstrate that α2 adrenergic agonists act directly on iHMGECs. However, these compounds do not elicit an overall negative effect. Rather, the α2 agonists promote the differentiation of iHMGECs.

Keywords: Brimonidine; Dry eye disease; Meibomian gland dysfunction; Phospholipidosis; α2 adrenergic agonist; α2 adrenergic antagonist.

Figures

Figure 1
Figure 1
Impact of brimonidine on proliferation and p-AKT signaling in iHMGECs. (A, B) Cells were cultured in KSFM with brimonidine for 7 days and counted with a hemocytometer. Cell rounding and detachment began to occur within 3 days in the 500 μg/ml brimonidine group. (C) Cells were grown in KSFM supplemented with EGF and BPE for 2 days, starved in KSFM overnight, and incubated with brimonidine for 15 minutes prior to lysis and immunoblotting. Band intensity was normalized to β-actin and analyzed using ImageJ. *P < 0.05 and **P < 0.005. Data are shown as mean ± standard error. One representative result of 3 total experiments is shown.
Figure 2
Figure 2
Effect of brimonidine, clonidine and phenylephrine on lysosome and neutral lipid accumulation in iHMGECs. Cells were treated in DMEM/F12 containing 10% FBS with or without drugs for 5-7 days. Cells were then stained for lysosomes (LysoTracker Red DND-99) and neutral lipids (HCS LipidTOX Green) and investigated under a fluorescent microscope. Azithromycin was used as a positive control. The results shown are from a single representative of 3 experiments. Scale bar indicates 50 μm.
Figure 3
Figure 3
Influence of brimonidine, clonidine and phenylephrine on phospholipid accumulation in iHMGECs. Cells were treated with or without α agonists in the presence of a phospholipidosis detection reagent for 6 days. Blue indicates lysosomes, green presents neutral lipids, and red demonstrates phospholipids. Azithromycin was used as a positive control. The results shown are from single representative of at least 2 experiments with each treatment. Scale bar indicates 50 μm.
Figure 4
Figure 4
Impact of brimonidine, clonidine and phenylephrine on signaling pathways. (A) Cells were maintained in DMEM/F12 containing 10% FBS for 6 days, serum-starved overnight, then treated with drugs for 15 minutes prior to lysis and immunoblot. Band intensity was normalized to the loading control (β-actin/ERK2/P38/JNK) and analyzed using ImageJ. (B) Cells were grown in KSFM overnight, stimulated with drugs for 15 minutes, lysed, and assayed for cAMP accumulation. *P < 0.05 and **P < 0.005. One representative result of 3 experiments is shown as mean ± standard error.
Figure 5
Figure 5
Effect of brimonidine on the expression of SREBP-1, LAMP-1 and LC3A in iHMGECs. Cells were treated with brimonidine for 6 days in DMEM/F12 containing 10% FBS and evaluated by immunoblot. Band intensity was normalized to β-actin and analyzed using ImageJ. One representative result of at least 2 experiments is shown as mean ± standard error. *P < 0.05 and **P < 0.005.
Figure 6
Figure 6
Influence of RX821002 and MK912 on lysosome and neutral lipid accumulation in iHMGECs. Cells were treated in DMEM/F12 containing 10% FBS with drugs for 5 (A, B) or 2 (C) days. Cells were stained with LysoTracker Red DND-99 and HCS LipidTOX Green neutral lipid for lysosomes and neutral lipids. Merged images show the co-localization of lysosome and neutral lipid. The results shown are from one of at least 2 experiments. Scale bar indicates 50 μm.

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