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. Author manuscript; available in PMC: 2019 May 1.
Published in final edited form as: Exp Eye Res. 2018 Feb 13;170:20–28. doi: 10.1016/j.exer.2018.02.009

Effect of brimonidine, an α2 adrenergic agonist, on human meibomian gland epithelial cells

Xi Han a,b, Yang Liu a, Wendy R Kam a, David A Sullivan a
PMCID: PMC5924632  NIHMSID: NIHMS945351  PMID: 29452108

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, phospholipidosis, α2 adrenergic agonist, α2 adrenergic antagonist, meibomian gland dysfunction, dry eye disease

1. Introduction

Recently, we 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) (Zhang et al., 2017). These effects are adverse, because HMGEC produce secretions that enhance tear film stability, prevent its evaporation, and promote ocular surface health (Knop et al, 2011). Interference with HMGEC function leads to meibomian gland dysfunction (MGD), destabilization of the tear film, and evaporative dry eye disease (DED) (Knop et al, 2011). DED, in turn, is characterized by tear film hyperosmolarity, ocular surface inflammation and damage, and pain (Craig et al, 2017).

It is possible that another class of anti-glaucoma drugs, the α2 adrenergic agonists, may also act directly on HMGECs to affect their structure and function, and lead to the development of MGD and DED. Our rationale is twofold. First, we have identified the mRNAs for α2 adrenergic receptors in mouse meibomian glands (Knop et al., 2011); and second, topical application of the α2 adrenergic agonist brimonidine has been shown to elicit foreign body sensations, visual blurring, ocular discomfort and DED (Fraunfelder et al., 2012; Hartleben et al., 2017; Schuman et al., 1997; Servat and Bernardino, 2011; Whitson et al., 2004, 2006). If α2 adrenergic agonists do induce MGD, this effect would be analogous to that of other anti-glaucoma drugs (Agnifili et al., 2013; Arita et al., 2012; Batra et al., 2014; Cunniffe et al., 2011; Custer and Kent, 2016; Mocan et al., 2016; Uzunosmanoglu et al., 2016), and help to explain further why topical anti-glaucoma treatment often aggravates DED and other ocular surface diseases (Fraunfelder et al., 2012; Servat and Bernardino, 2011; Uzunosmanoglu et al., 2016; Gomes et al., 2017).

The purpose of this investigation was to test our hypothesis that α2 adrenergic agonists act directly on HMGECs to influence their structure and function. To represent this class of drugs, we chose brimonidine and focused on its effect on the morphology, survival, proliferation and differentiation of HMGECs. We also examined the chemical specificity of brimonidine’s action, and the mechanism(s) underlying this compound’s influence on these cells.

2. Materials and Methods

2.1 Drugs

Brimonidine (α2 agonist), clonidine (α2 agonist) and phenylephrine (α1 agonist) were purchased from Cayman Chemical (Ann Arbor, Michigan, USA) and dissolved in keratinocyte serum-free medium (KSFM; Thermo-Fisher Scientific, Grand Island, NY, USA) to prepare 5 mg/ml stocks. Brimonidine concentrations were chosen based upon the topical clinical dose (i.e. 0.2%, which equals 2 mg/ml), as well as studies investigating the levels (i.e. ~ 50 μg/ml) of brimonidine in the conjunctiva after topical administration (Acheampong et al., 2002). The α2 antagonists RX821002 and MK912 (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 (DMEM/F12; Corning Cellgro, Manassas, VA, USA) containing 10% fetal bovine serum (FBS; Thermo-Fisher Scientific, Grand Island, NY, USA) to make 10 mg/ml and 2 mg/ml stocks, respectively. Forskolin and 3-isobutyl-1-methylxanthine (IBMX, both Sigma-Aldrich) were dissolved in ethanol to form 10 mM and 45 mM stocks, respectively. Stocks were further diluted in culture media to the concentrations described.

2.2 Cell Proliferation Assay

Immortalized HMGECs (iHMGECs), which were developed in our laboratory with retroviral human telomerase reverse transcriptase (Liu et al., 2010), were seeded in 12 well plates (3×104 cell / well) in KSFM with or without brimonidine for 7 days. KSFM supplemented with 5 ng/ml epidermal growth factor (EGF) and 50 μg/ml bovine pituitary extract (BPE) (Thermo-Fisher Scientific), which are known to promote proliferation of iHMGECs, was used as a positive control (Zhang et al., 2017). Cellular morphology was recorded and cells were counted with a hemocytometer. Experiments were performed in triplicate and repeated at least 3 times.

2.3 Cellular staining

Cells were treated in DMEM/F12 containing 10% FBS in the presence or absence of HCS LipidTOX™ Red phospholipidosis detection reagent for 5-7 days. Cells were stained for lysosome accumulation using LysoTracker® Red DND-99 or LysoTracker® Blue DND-22 and for neutral lipid with HCS LipidTOX™ Green neutral lipid stain (Zhang et al., 2017). All stains were purchased from Thermo-Fisher Scientific. Cells treated with 10 μg/ml azithromycin (Santa Cruz Biotechnology, Dallas, TX, USA) were used as a positive control for differentiation (Zhang et al., 2017). Each experiment was performed in duplicate and four random pictures were taken of each sample. Experiments were performed at least twice.

2.4 SDS-PAGE and Western blots

In order to investigate signaling pathways in proliferating cells, iHMGECs were maintained in KSFM supplemented with EGF and BPE for 2 days and starved in KSFM overnight. To identify changes to iHMGECs in differentiating conditions, cells were exposed to DMEM/F12 containing 10% FBS for 6 days. In signal pathway experiments, cells were starved overnight in DMEM/F12 containing 1% FBS and then treated with drugs for 15 minutes. In other experiments, cells were grown in DMEM/F12 containing 10% FBS with brimonidine for 6 days prior to lysis and immunoblot as previously described (Zhang et al., 2017). Primary antibodies were specific for phosphorylated phosphoinositide 3-kinase-protein kinase B (p-Akt, 1:4000, Cell Signaling Technology, Danvers, MA, USA), phosphorylated extracellular signal-regulated kinase (p-ERK, 1:2000, Santa Cruz Biotechnology), ERK2 (1:4000, Santa Cruz Biotechnology), phosphorylated p38 mitogen activated protein kinase (p-p38, 1:1000, Cell Signaling Technology), p38 (1:1000, Cell Signaling Technology), phosphorylated c-Jun N-terminal kinases (p-JNK, 1:2000, Santa Cruz Biotechnology), JNK (1:1000, Santa Cruz Biotechnology), sterol regulatory element-binding protein 1 (SREBP-1, H-160, 1:500, Santa Cruz Biotechnology), light chain 3A (LC3A, 1:1000, Cell Signaling Technology), lysosomal-associated membrane protein 1 (LAMP-1, H4A3-s, 1:500, Developmental Studies Hybridoma Bank, Iowa City, IA) and β-actin (1:10,000, Cell Signaling Technology). Densitometry was performed with publicly available image-processing software (ImageJ; http://rsb.info.nih.gov/ij). Experiments were performed in triplicate and repeated at least twice.

2.5 cAMP ELISA

Cells were plated in 96 well plates at a density of 5×104 cells / well overnight, then treated with brimonidine, forskolin, or IBMX, alone or in combination, for 15 minutes. Cellular adenosine 3,5-cyclic monophosphate (cAMP) was measured using the Amersham cAMP Biotrak Enzymeimmunoassay (EIA) System (GE Health Care, Buckinghamshire, United Kingdom), according to the nonacetylation protocol supplied by the manufacturer. Forskolin, an activator of adenylate cyclase, and IBMX, a competitive inhibitor of phosphodiesterase, were used as positive controls for cAMP accumulation (Essayn, 2001). A standard curve in duplicate was run in each assay. Three independent experiments in triplicate were conducted.

2.6 Statistics

We performed one-way analysis of variance and unpaired, 2-tailed Student’s t-tests. All statistical analyses were performed using commercially available software (Prism 5; GraphPad Software, Inc, La Jolla, CA, USA).

3. Results

3.1 Influence of brimonidine on the morphology, survival and proliferation of iHMGECs

To evaluate the effect of brimonidine on the proliferation of iHMGECs, cells were treated with various doses of this compound (i.e. 500 μg/ml, 50 μg/ml or 5 μg/ml) or vehicle and observed for seven days. These brimonidine concentrations were equivalent to one-fourth the clinical dose of 2 mg/ml, the conjunctival level (i.e. physiological) of ~ 50 μg/ml that follows topical administration (Acheampong et al., 2002), and one-tenth the latter amount. It is likely that HMGECs in vivo are exposed to the “physiological” concentration.

As illustrated in Figure 1, exposure to 500 μg/ml brimonidine induced cell rounding and detachment within three days, and a significant reduction in cell number. In contrast, neither the 50 μg/ml nor the 5 μg/ml brimonidine concentrations had any effect on iHMGEC morphology or number (Figure 1 A, B).

Figure 1.

Figure 1

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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.

Given the impact of high dose brimonidine on iHMGEC survival, we examined whether this compound has a dose-dependent effect on the Akt signaling pathway. Akt promotes cell survival, growth and proliferation (Jin et al., 2012). As demonstrated in Figure 1C, the 500 μg/ml brimonidine treatment, but not the other brimonidine concentrations, significantly decreased p-Akt levels.

3.2 Effects of brimonidine, clonidine and phenylephrine on the differentiation of iHMGECs

To determine whether brimonidine influences the differentiation of iHMGECs, we cultured cells for 5–7 days in DMEM/F12 supplemented with 10% FBS, and vehicle, or 500 μg/ml, 50 μg/ml or 5 μg/ml of brimonidine, or azithromycin (10 μg/ml) as a positive control. We then stained cells for the analysis of lysosomes and neutral lipids. As demonstrated in Figure 2 A, the 500 μg/ml and 50 μg/ml concentrations of brimonidine, but not the 5 μg/ml level, stimulated a pronounced accumulation of lysosomes and neutral lipids in iHMGECs. The neutral lipids co-localized within lysosomes, in a manner analogous to that following azithromycin treatment (Figure 2A, “Merge”).

Figure 2.

Figure 2

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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.

To examine the adrenergic specificity of brimonidine’s differentiative influence, we cultured iHMGECs with clonidine, another α2 adrenergic agonist, and phenylephrine, an α1 adrenergic agonist, for 7 days. Initially, we chose clonidine concentrations of 500 μg/ml and 50 μg/ml to match those of brimonidine that stimulated cell differentiation. However, the 500 μg/ml clonidine dose was toxic (data not shown), and consequently we reduced the upper clonidine level to 200 μg/ml. As illustrated in Figure 2B, the 200 μg/ml and 50 μg/ml clonidine treatments duplicated brimonidine’s effect on the accumulation of lysosomes and neutral lipids. In contrast, iHMGEC exposure to 500 μg/ml, 50 μg/ml or 5 μg/ml of phenylephrine had no impact on these cellular parameters (Figure 2 C).

3.3 Impact of brimonidine, clonidine and phenylephrine on phospholipid accumulation in iHMGECs

We have previously discovered that azithromycin, a potent phospholipidosis (PLD)-inducing cationic amphiphilic drug (CAD) (Ribeiro et al., 2009; Tyteca et al., 2001), elicits a PLD-like effect in iHMGECs (Liu et al., 2014). Thus, azithromycin promotes the accumulation of phospholipids, cholesterol and lysosomal lamellar bodies in iHMGECs (Liu et al., 2014). Given that the intracellular accumulation of phospholipids in lysosomes is one of the hallmark features of PLD (Reasor and Kacew, 2001; Sadrieh, 2010), we sought to determine whether brimonidine and clonidine also induce a PLD-like effect in iHMGECs. For comparison, we tested the influence of phenylephrine as well.

As shown in Figure 3, brimonidine at a concentration of 500 μg/ml, and clonidine at 200 μg/ml, both stimulated phospholipid accumulation in iHMGEC lysosomes. Their PLD-like effect was similar to that of azithromycin (Figure 3). Phenylephrine at 500 μg/ml had no demonstrable influence on phospholipid appearance (Figure 3).

Figure 3.

Figure 3

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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.

3.4 Effects of brimonidine, clonidine and phenylephrine on signaling pathways in iHMGECs

To determine whether the α2 adrenergic agonist induction of iHMGEC differentiation involves the activity of signaling pathways, we evaluated the ability of 500 μg/ml brimonidine, 200 μg/ml clonidine and 500 μg/ml phenylephrine to modulate the levels of Akt, ERK, p38 and JNK, with or without phosphorylation. These pathways have all been linked to α2 adrenergic receptors (Caine, 1998; Kajiya et al., 2012; Kobayashi et al., 2007).

Our findings demonstrate that all three adrenergic agonists decreased p-Akt expression, as compared to that of the vehicle control (Figure 4A). Brimonidine and clonidine also reduced p-p38 levels, whereas only phenylephrine suppressed p-ERK (Figure 4A). None of the drugs altered p-JNK.

Figure 4.

Figure 4

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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.

As part of these analyses we tested whether 500 μg/ml brimonidine exposure, in the presence or absence of forskolin (10−6 M) or IBMX (1 mM), influences cAMP levels in iHMGECs. Our data show that both forskolin and IBMX stimulate cAMP accumulation, but that brimonidine had no effect (Figure 4B). Given this observation, we did not examine the comparative influence of either clonidine or phenylephrine on the adenylate cyclase pathway.

3.5 Influence of brimonidine on the expression of protein biomarkers of differentiation in iHMGECs

We have previously discovered that the differentiation of primary and immortalized HMGECs is associated with an upregulation of genes linked to lysosomes, lipids, phagosomes and autophagy (Sullivan et al., 2014). To assess whether brimonidine’s influence on iHMGEC differentiation involves the translation of such genes, we treated cells with brimonidine for 6 days and analyzed cell lysates for SREBP-1, LAMP-1 and LC3A. SREBP-1 is a key lipogenesis regulator, which increases the synthesis of enzymes involved in fatty acid, lipid and cholesterol production (Eberle et al., 2004). SREBP-1 is activated by cleavage of a precursor into the mature form (Horton et al., 2002), resulting in a cluster of protein bands between 59 and 68 kDa (Gosmain et al., 2005). LAMP-1 and LC3 are biomarkers for lysosomes and autophagosomes, respectively (Eskelinen, 2006; Tanida et al., 2008).

As shown in Figure 5A, treatment with 500 μg/ml brimonidine increased the mature, and decreased the precursor, forms of SREBP-1, indicating that brimonidine promotes the conversion of SREBP-1 to the active moiety. Brimonidine also enhanced the expression of both LAMP-1 and LC3A (Figure 5B).

Figure 5.

Figure 5

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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.

3.6 Effect of α2 adrenergic antagonists on the brimonidine-induced differentiation of iHMGECs

To determine whether α2 adrenergic receptor antagonists interfere with brimonidine-induced differentiation of iHMGECs, we cultured cells for 5 days with vehicle, azithromycin (10 μg/ml, positive control), or RX821002 (1,000 μg/ml) or MK-912 (1,000 μg/ml) in the presence or absence of brimonidine (500 μg/ml). RX821002 demonstrates inverse agonist properties at α2 adrenergic receptors (Clarke and Harris, 2002, Murrin et al., 2000), which means that it should induce a pharmacological responseopposite to that of brimonidine. MK-912 acts as a neutral agonist (Murrin et al., 2000), and should have no activity in the absence of brimonidine, but be able to inhibit brimonidine action. We selected antagonist concentrations based upon reported binding affinities for α2A adrenergic receptor subtype (Table 1) (Lomasney et al., 1991, MacDonald et al., 1997, Uhlen et al., 1995).

Table 1.

Affinities of α adrenergic agonists and antagonists for α adrenergic receptor subtypes

Compound Receptor binding affinity (Ki, nM)
α2A α2B α2C
α2 Adrenergic agonist
 Brimonidine 3.7 ± 0.8 512 ± 3 120 ± 13
 Clonidine 11 ± 3 40 ± 4 134 ± 4
α2 Adrenergic antagonist
 RX821002 0.14 ± 0.01 7.9 ± 0.8 1.1 ± 0.1
 MK912 0.40 ± 0.03 8.3 ± 0.4 0.08 ± 0.01
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The α2A, α2B and α2C are adrenergic receptor subtypes. The Ki is an inhibitor constant, and equal to the concentration necessary to produce half maximum inhibition. The listed concentrations are from Uhlen et al. [44] and Lomasney et al. [45]. For comparison, phenylephrine has affinities for α1A (1,400), α1B (23,900) and α1C (47,800) receptors [46].

Our results show that neither RX821002 nor MK-912 acted as overall antagonists of brimonidine-induced differentiation of iHMGECs (Figures 6A, B). Indeed, both compounds stimulated the accumulation of lysosomes and/or neutral lipids. At the RX821002 and MK-912 concentrations used, though, as with brimonidine at 500 μg/ml, there were relatively few cells remaining after 5 days of culture. Consequently, we reduced the concentrations of RX821002 and MK-912 to 100 and 200 μg/ml and examined their effect on iHMGECs after 2 days of culture. As illustrated in Figure 6C, both RX821002 and MK-912 acted like α2 adrenergic receptor agonists and increased the iHMGEC content of lysosomes and neutral lipids.

Figure 6.

Figure 6

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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.

4. Discussion

Our results show that brimondine exposure induces a dose-dependent reduction in iHMGEC Akt signaling and proliferation. In contrast, brimonidine promotes a dose-dependent differentiation of iHMGECs, including an increase in neutral lipid, phospholipid and lysosome levels. These effects were associated with an inhibition of p38 signaling, and were reproduced by cellular exposure to clonidine, but not phenylephrine. Brimonidine also enhanced the cellular content of SREBP, LAMP-1 and LCA3. Of particular interest, the putative α2 antagonists, RX821002 and MK912, did not inhibit brimonidine action, but rather stimulated iHMGEC differentiation. Overall, our findings support our hypothesis and demonstrate that α2 adrenergic agonists act directly on iHMGECs. However, unlike other anti-glaucoma pharmaceuticals (e.g. timolol and pilocarpine), these α2 adrenergic drugs do not cause a general negative effect. Rather, the α2 agonists stimulate iHMGEC differentiation.

Topical clonidine was the first α2 adrenergic agonist to be marketed for the treatment of glaucoma, but brimonidine is more widely used because of its higher α2 adrenergic receptor selectivity and lower systemic side effects (Apatachioae and Chiselita, 1999). The α1 agonist, phenylephrine, does not have a role in glaucoma therapy. The stimulation of iHMGEC lipid production by brimonidine and clonidine, but not by phenylephrine, suggests that α2, and not α1, adrenergic receptor agonists alter the activity of one or more signaling pathways that promote differentiation. One such pathway could be that of p38. We found that brimonidine and clonidine, but not phenylephrine, decreased the phosphorylation of p38. This response may facilitate lipid accumulation, because p38 typically suppresses lipid synthesis. Thus, a reduction in p38 activity is paralleled by a rise in liver fat levels (Xiong et al., 2007), as well as an increase in adipogenesis (He et al., 2013). Whether a specific link exists between p38 suppression and lipid accrual in HMGECs, though, remains to be determined.

We discovered that brimonidine and clonidine stimulate phospholipid accumulation in iHMGEC lysosomes, and that this PLD-like effect is analogous to that caused by azithromycin. This finding suggests that these α2 adrenergic agonists may be CADs. In support of this hypothesis, we observed the change in phospholipid dynamics by using the LipidTOX Red reagent, which has been utilized extensively to identify CAD-induced PLD (Shahane et al., 2014). In addition, brimonidine, like CADs, is positively charged at physiological pH (Ueda et al., 2000), and could incorporate, like CADs, into the negatively charged lysosomal membranes Muehlbacher et al., 2012). Further, clonidine has been postulated to be a CAD (Kubo, 2016). If brimonidine and clonidine are CADs, they may act through multiple possible mechanisms to promote PLD. These include stimulating the synthesis, and/or preventing the degradation, of phospholipids (Halliwell 1997, Muehlbacher et al., 2012).

To begin to understand the mechanism of lipid synthesis in iHMGECs in response to brimonidine treatment, we investigated the expression of three relevant proteins. Brimonidine elicits upregulation of SREBP-1, LAMP-1 and LC3A in iHMGECs. SREBP-1, a master regulator of lipid synthesis (Eberle et al., 2004), is synthesized as an inactive precursor and cleaved into an active mature form. Brimonidine triggers the conversion of the SREBP-1 precursor to the mature form, promoting lipid accumulation in iHMGECs. LAMP-1 is a transmembrane protein, which resides primarily across lysosomal membranes (Carlsson and Fukuda, 1989). Brimonidine stimulation results in a dose-dependent upregulation of LAMP-1, which is consistent with increased lysosome staining. Of particular note, brimonidine at high concentration contributes to a prominent increase of LC3A, one isoform of LC3, which is the most widely-monitored autophagy-related protein (Klionsky et al., 2016). At a physiological concentration, brimonidine promotes iHMGEC differentiation (more accumulation of neutral lipids and lysosomes) but not autophagy (no upregulation of LC3A). However, at a high concentration, brimonidine induces both terminal differentiation (more striking neutral lipid and lysosomal accumulation) and autophagy (obvious upregulation of LC3A), suggesting a role for autophagy in terminal differentiation in the meibomian gland (Fischer et al., 2017).

To our surprise, the two α2 adrenergic antagonists, RX821002 and MK912, did not inhibit, but rather duplicated, the differentiative effects of brimonidine and clonidine on iHMGECs. We are unaware of other studies reporting α2 adrenergic agonist activity for RX821002 and MK912. However, such an action is not unique. The selective estrogen receptor modulators, tamoxifen, raloxifene, and toremifen, all exhibit mixed agonist and antagonist ability, depending on the target tissue (Cosman and Lindsay, 1999; Gallo and Kaufman, 1997). Opioids are also known to act as agonists or antagonists, depending upon the receptor type (Hoskin and Hanks, 1991). It may be that the iHMGEC differentiation reflects a cell-specific response to RX821002 and MK912.

Given our discovery that brimonidine stimulates iHMGEC differentiation, how then does the topical application of brimonidine elicit foreign body sensations, visual blurring, ocular discomfort and DED (Fraunfelder et al., 2012; Hartleben et al., 2017; Schuman et al., 1997; Servat and Bernardino, 2011; Whitson et al., 2004, 2006)? These adverse events may be related to the corneal toxicity (Robciuc et al., 2017), goblet cell loss (Aydin et al., 2014), and conjunctival hyperaemia and inflammation (Hartleben et al., 2017 and Serle, 1996) associated with brimonidine adminstration.

Our goal is to determine whether brimonidine is effective in vivo by using a mouse model of obstructive MGD. This model, which was developed to facilitate understanding of MGD pathophysiology, is generated by feeding HR-1 hairless mice a special diet with limited lipid content (Miyake et al., 2016). These mice develop markedly plugged (i.e. obstructed) meibomian gland orifices, hyperkeratinization of the terminal duct epithelium and toothpaste-like meibomian gland secretions. Hyperkeratinization of the ductal epithelium and reduced meibum quality, in turn, contribute significantly to the luminal plugging found in human obstructive MGD (Knop et al., 2011). Moreover, topical application of azithromycin, a CAD, ameliorates the MGD in this model (Miyake et al., 2016). Hence, this mouse model, which mimics many of the characteristics of human MGD, may well be responsive to brimonidine therapy.

Overall, unlike other anti-glaucoma drugs that induce MGD (Agnifili et al., 2013; Arita et al., 2012; Batra et al., 2014; Cunniffe et al., 2011; Custer and Kent, 2016; Mocan et al., 2016; Uzunosmanoglu et al., 2016), our findings suggest that α2 adrenergic agonists, especially at physiological levels, are beneficial for HMGEC function.

Highlights.

  • Brimonidine stimulates the differentiation of human meibomian gland epithelial cells

  • Brimonidine decreases the proliferation of, and Akt and p38 signaling in, these cells

  • Brimonidine’s cellular effects are duplicated by clonidine, but not by phenylephrine

  • Brimonidine enhances the cellular content of sterol regulatory binding protein-1

Acknowledgments

Funding

This work was supported by NIH grant EY028653 and the Margaret S. Sinon Scholar in Ocular Surface Research Fund.

Abbreviations

AKT

phosphoinositide 3-kinase-protein kinase B

BPE

bovine pituitary extract

CAD

cationic amphiphilic drug

cAMP

adenosine 3,5-cyclic monophosphate

DED

dry eye disease

DMEM/F12

1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12

EGF

epidermal growth factor

ERK

extracellular signal-regulated kinase

FBS

fetal bovine serum

IBMX

3-isobutyl-1-methylxanthine

iHMGECs

immortalized human meibomian gland epithelial cells

JNK

c-Jun N-terminal kinases

KSFM

keratinocyte serum-free medium

LAMP-1

lysosomal-associated membrane protein 1

LC3A

light chain 3A

MGD

Meibomian gland dysfunction

p38

p38 mitogen activated protein kinase

SREBP-1

sterol regulatory element-binding protein 1

Footnotes

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