brimonidine can reverse in vitro hydroquinone damage on retinal pigment epithelium cells
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Brimonidine Can Reverse in vitro Hydroquinone Damage on
Retinal Pigment Epithelium (RPE) Cells and Retinal Müller
Cells (MIO-M1)
Journal: Journal of Ocular Pharmacology and Therapeutics
Manuscript ID: JOP-2015-0083
Manuscript Type: Original Article
Date Submitted by the Author: 02-Jul-2015
Complete List of Authors: Ramírez, Claudio; Gavin Herbert Eye Institute, University of California Irvine, Caceres del Carpio, Javier; Gavin Herbert Eye Institute, University of California Irvine, Chu, Justin; Gavin Herbert Eye Institute, University of California Irvine, Chu, Joshua; Gavin Herbert Eye Institute, University of California Irvine, Moustafa, M. Tarek; Gavin Herbert Eye Institute, University of California Irvine, ; Minia University, Ophthalmology Chwa, Marilyn; Gavin Herbert Eye Institute, University of California Irvine, Limb, G. Astrid; UCL Institute of Ophthalmology, Division of Ocular Biology and Therapeutics Kuppermann, Baruch; Gavin Herbert Eye Institute, University of California Irvine, Kenney, M. Cristina; Gavin Herbert Eye Institute, University of California Irvine,
Keywords: Neuroprotection, Retinal Piment Epithelium, Muller Cell
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
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Brimonidine Can Reverse in vitro Hydroquinone Damage on Retinal
Pigment Epithelium (RPE) Cells and Retinal Müller Cells (MIO-M1)
Claudio Ramírez1*
, Javier Cáceres-del-Carpio
1*, Justin Chu
1, Joshua Chu
1, M. Tarek
Moustafa1,2
, Marilyn Chwa1, G. Astrid Limb
3, Baruch D. Kuppermann
1, M. Cristina
Kenney1,4**
1 Gavin Herbert Eye Institute, University of California, Irvine, California, USA
2 Ophthalmology Department, Minia University, Egypt
3 Division of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, UK
4 Department of Pathology and Laboratory Medicine, Univ. of California Irvine, Irvine, CA;
* Authors contributed equally to this manuscript.
** Corresponding author
Keywords: Brimonidine, Hydroquinone, Cell Viability, Müller Cells, Retinal Pigment Epithelial
Cells, Neuroprotection
Abbreviated Title: Brimonidine protects ARPE-19 and MIO-M1 cells
Address Correspondence To:
M. Cristina Kenney, M.D., Ph.D.
Gavin Herbert Eye Institute,
843 Health Science Road,
University of California Irvine, CA
Telephone: (949) 824-7603,
Fax: (949) 824- 9626,
Email: [email protected]
Supported by the Discovery Eye Foundation, Iris and B. Gerald Cantor Foundation, Henry L.
Guenther Foundation, Polly and Michael Smith Foundation, Lincy Foundation
JCdC is the David and Julianna Pyott Pan-American Retina Research Fellow
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Authors:
Claudio Ramírez*
Gavin Herbert Eye Institute, University of California, Irvine
843 Health Sciences Rd., Irvine CA 92697
Javier Cáceres-del-Carpio*
Gavin Herbert Eye Institute, University of California, Irvine
843 Health Sciences Rd., Irvine CA 92697
Justin Chu
Gavin Herbert Eye Institute, University of California, Irvine
843 Health Sciences Rd., Irvine CA 92697
Joshua Chu
Gavin Herbert Eye Institute, University of California, Irvine
843 Health Sciences Rd., Irvine CA 92697
M. Tarek Moustafa
Gavin Herbert Eye Institute, University of California, Irvine
843 Health Sciences Rd., Irvine CA 92697
Ophthalmology Department, Minia University, Egypt
Corniche El Nil, Minia University Hospital, Minia, Egypt 61111
Marilyn Chwa
Gavin Herbert Eye Institute, University of California, Irvine
843 Health Sciences Rd., Irvine CA 92697
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G. Astrid Limb
Division of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology,
11-43 Bath Street, London. EC1V 9EL, United Kingdom
Baruch D. Kuppermann
Gavin Herbert Eye Institute, University of California, Irvine
850 Health Sciences Rd., Irvine CA 92697
M. Cristina Kenney**
Gavin Herbert Eye Institute, University of California, Irvine
843 Health Sciences Rd., Irvine CA 92697
* Authors contributed equally to this manuscript.
** Corresponding author
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ABSTRACT
Purpose: Brimonidine is a selective alpha-2 adrenergic agonist used to reduce intraocular pressure
and it has been shown to have some neuroprotective effects. Hydroquinone (HQ) is a toxicant present
in cigarette smoke, and other sources. In this study, we investigated the cyto-protective effects in
vitro of Brimonidine on human retinal pigment epithelium cells (ARPE-19) and human retinal Müller
cells (MIO-M1) that had been treated with HQ.
Methods: Cells were pre–treated for 6 hours with different doses of Brimonidine tartrate 0.1% (½x,
1x, 5x, 10x), followed by a 24-hour exposure to 100 µM of HQ, while the Brimonidine was still
present. Assays were used to measure cell viability, mitochondrial membrane potential (∆Ψm),
reactive oxygen species (ROS) production, and lactate dehydrogenase (LDH) release.
Results: Brimonidine increased the cell viability at all concentrations studied in both cell lines
studied. ∆Ψm also improved at all Brimonidine doses in ARPE-19 cells and in the 5x and 10x
dosages MIO-M1 cells. The ROS levels decreased at 1x, 5x and 10x doses of Brimonidine in ARPE-
19 but only at 10x on MIO-M1 cells. The 10x-Brimonidine ARPE-19 cells had decreased LDH
release, but no DHL changes were observed on MIO-M1 cells.
Conclusion: HQ-induced toxicity is mediated through mitochondrial-damaging, oxidative stress-
related and necrosis-related pathways; Brimonidine significantly reversed the mitochondrial-
damaging and oxidative stress-related effects but had little effect on blocking the necrosis component
of HQ-toxicity. Brimonidine protective effects differ between the different retinal cell types and high
concentrations of Brimonidine (10x) have minimal damaging effects on human retinal cells.
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INTRODUCTION
Brimonidine is a selective alpha-2 adrenergic agonist, commonly used topically to lower the
intraocular pressure (IOP) in primary open-angle glaucoma (POAG) and ocular hypertension. The
mechanisms of action for alpha-2 agonists are to inhibit adenylate cyclase, decrease intracellular
cAMP and cause ciliary vasoconstriction, which reduces aqueous production in the ciliary body and
increases uveoscleral outflow, leading to IOP reduction.1-3
Several studies have proved the safety and efficacy of Brimonidine alone or combined with other
glaucoma treatments in lowering intraocular pressure.1, 4
Optic neuroprotection is commonly referred
to the protection of retinal ganglion cells (RGC) from different optic nerve injuries.5 Authors have
proposed that to be considered neuroprotective, a drug should accomplish four criteria: (1) It must
target receptors on its target tissue; (2) It must show pharmacological levels of penetration into the
vitreous and retina; (3), It must induce intracellular changes in neurons that impede apoptosis or
increase neuronal resistance to the insult; and (4) similar efficacy must be shown in clinical trials.6
Several in vitro and animal models studies have suggested a neuroprotective effect of Brimonidine to
different optic nerve insults, including glaucomatous optic neuropathy, ischemic neuropathies, and
other optic nerve disorders.7-9
Brimonidine has also been suggested to protect photoreceptors from
phototoxicity.10
Brimonidine transport has been described in retinal pigment epithelium (RPE) cells
in vitro and in animal models 11
; and alpha-2 agonist receptors have been found in Müller cells in
vitro and in vivo.12
Furthermore, in increased IOP and normal tension glaucoma animal models, a
role of Müller cells in neuroprotection of retinal ganglion cells has been suggested.13, 14
Smoking is an important risk factor for age-related macular degeneration (AMD).15
In 2000, the Age-
related Eye Disease Study (AREDS), a large epidemiologic study, found association between
smoking and advanced AMD, including geographic atrophy and neovascularization.16
The 10-year
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follow up of the initial AREDS subjects, showed that current smoking is also a risk factor for
progression to neovascular AMD.17
Oxidative stress and inflammation are believed to be possible
mechanisms for this association.18, 19
Cigarette smoke contains thousands of toxic and carcinogenic
chemicals,20, 21
which can be divided into two categories: the gas-phase smoke components and
substances retained in the filter (referred to tar). The most common component in cigarette tar is
hydroquinone (HQ), semiquinone mixture.22
HQ is also used as a reducing agent in the
petrochemical, rubber and polymers industries. HQ is found in black and white photography paper
and is used in x-ray film processing.23
Topical cream HQ formulations are commercialized for
bleaching hyperpigmented skin, but with concerns from Food and Drug Administration (FDA).24
Previous in vitro studies with retinal cells have shown toxicity from HQ, nicotine and benzo(e)pyrene
(BeP), each a key cigarette smoke compound.25-29
HQ has also been found in vitro and in animal
models to affect gene expression of oxidative stress related mediators and genes related to AMD
pathogenesis.30-32
HQ can reduce the activity of matrix metalloproteinase 2 (MMP-2) in vitro 30
,
down regulate the monocyte chemoattractant protein 1 (MCP1), upregulate VEGF and PEDF
expression in vitro and mice31
; and upregulate the expression of heat shock protein 27 (Hsp-27) in
mice and in vitro models.32
The human retina integrates different types of cells and neurons with the photoreceptors. The retinal
pigment epithelium (RPE), which overlies Bruch’s membrane, is located between the retina and
choriocapillaris and constitutes the retinal-blood barrier. The RPE functions include light absorption
and protection to photo-oxidation, transepithelial transport between choriocapillaris and
photoreceptors, ion buffering and homeostasis in the subretinal space, phagocytosis of the outer
segments of photoreceptors, and secretion of platelet derived growth factor (PDGF) and vascular
endothelial growth factor (VEGF).33
Loss of RPE cells is one of the earliest changes in AMD.
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Müller cells, the most common glial cell in the retina, provide structural and metabolic support to
retinal neurons, by regulating cellular homeostasis, including pH, and modulating neurotransmitter
recycling.34, 35
They also contribute to the retinal blood barrier by surrounding retinal capillaries with
glial processes 34, 35
and act as light collectors by directing light to photoreceptors.35, 36
Müller cells
secrete and regulate VEGF and PEDF, playing a role in wound healing, retinal regeneration and
neuroprotection.34, 35, 37
The purpose of this study was to evaluate the in vitro protective effects of different Brimonidine
concentrations on an immortalized cell line of human RPE cells (ARPE-19) and an immortalized cell
line of retinal Müller cells (MIO-M1) treated with HQ.
MATERIALS AND METHODS
Cell Culture
ARPE-19 cells were obtained from ATCC, Manassas, VA and then cultured in Dulbecco’s modified
Eagle’s medium containing 10 % fetal bovine serum, penicillin G 100 U/ml, streptomycin sulfate
0.1% mg/ml, gentamicin 10 µg/ml and amphotericin B 2.5 µg/ml. Serum free medium was used after
cells reached monolayer confluence. Human retinal Müller cells (MIO-M1), obtained from the
Department of Cell Biology of the University College, London 34
, were grown in a 1:1 mixture
(vol/vol) of Dulbecco’s Modified Medium High Glucose 1x (DMEM + GlutaMAX, Gibco, Carlsbad,
CA). Initially, cells were cultured in 10% FBS and penicillin G 100 U/ml, streptomycin sulfate 0.1
mg/ml, but once established, the culture media were changed to 2% FBS. This simulates the natural
human Müller cells to remain in a non-proliferating phase.
Pretreatment with Brimonidine
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Brimonidine tartrate 0.1% was obtained from an ophthalmic solution (Alphagan P, Allergan, Irvine,
CA) containing Purite as a vehicle. Several dilutions were made to reach four different
concentrations, 1x (which is defined as the clinical dose 50 µl in 4 ml of solution), ½x, 5x and 10x.
The highest concentration (10x) of Brimonidine alone, only dissolved in dimethyl sulfoxide (DMSO)
without HQ, was used as a control.
Exposure to HQ
Commercially available HQ powder was solubilized into DMSO and 100 µM solution prepared.
This concentration of HQ was taken from previous studies showing that this concentration could
stress the cells without being toxic.25, 26
Cells were pretreated with Brimonidine in the different
concentrations for 6 hours. Then cells were exposed to 100µM HQ in culture media and incubated
at 37ºC for 24 hours. Brimonidine was kept in the medium throughout the culture period.
Equivalent amount of DMSO was used as a control.
Controls
Three different controls were used in this study: 1) cells treated with 10x Brimonidine alone, 2)
cells treated with 100 µM DMSO (the maximum amount of DMSO used to solubilize the HQ) and
3) untreated cells.
Cell Viability Assay (CV)
5 x 105 cells per well were plated in six-well plates for 24 hours and treated as mentioned above.
Cells were harvested using trypsin-EDTA 0.2% for 5 minutes. Cells were centrifuged for 5 minutes
at 1000 rpm, and then resuspended in 1 ml of culture medium. Cell viability was assessed by trypan
blue dye-exclusion with an automated ViCell cell viability analyzer (Beckman Coulter Inc.,
Fullerton, CA).
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Mitochondrial membrane potential (∆Ψm)
Detection of ∆Ψm values was performed using the JC-1 mitochondrial membrane potential detection
kit (Biotium, Hayward, CA). JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-
carbocyanine-iodide) is a cationic dye that accumulates in the mitochondrial membranes of healthy
cells resulting in red fluorescence (590 nm). Stressed or damaged cells have reduced ∆Ψm and the
JC-1 dye accumulates in the cytosol instead of the mitochondria, resulting in green fluorescence
(530nm). The ratio of red to green fluorescence is calculated to obtain the changes in ∆Ψm. The
fluorescent signals were measured with the fluorescent image scanning unit FMBio III (Hitachi
Solutions America, San Bruno, CA) set to detect green (excitation (EX) 485 nm, emission (EM) 535
nm) and red (EX 550 nm, EM 600 nm) emissions.
ROS/RNS Assay
ROS/RNS production was assessed using the fluorescent dye
2',7'-dichlorodihydrofluorescein
diacetate (H2DCFDA; Invitrogen-Molecular Probes, Eugene, OR).
38 The fluorescent signal was
measured using the fluorescent image scanning unit FMBio III (Hitachi Solutions America, San
Bruno, CA) with EX filter in 550 nm and EM filter in 600 nm. This assay detects hydrogen
peroxide, peroxyl radicals, and peroxynitrite anions.
LDH (Lactate Dehydrogenase) Cytotoxicity Assay
The LDH assay was performed using the LDH Cytotoxicity Assay Kit II (BioVision, Inc., Mountain
View, CA) according to the manufacturer’s protocol. The amount of LDH released by the cells can
be used as an index of necrosis. This kit uses the WST ((4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-
5-tetrazolio]-1,3-benzene disulfonate) reagent for detection of LDH released from the damaged
cells. LDH oxidizes lactate to generate NADH, reacting with WST, which can be quantified at
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450nm optical density (OD) with a BioTek ELx808 absorbance plate reader (BioTek, Winooski,
VT).
Statistical Analyses
Data were analyzed with unpaired t-test and one-way ANOVA using the GraphPad Prism version
5.00 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com). P < 0.05 (*)
statistically significant; P < 0.01 (**) very significant; P < 0.001 (***) extremely significant. Error
bars in the graphs represent SEM with experiments performed in triplicate.
RESULTS
Cell Viability Studies
Mean percentage cell viability for ARPE-19 cells exposed to 100 µM of HQ was 27.92 ± 0.66
compared to the DMSO-equivalent of 95.73 ± 0.84 (P < 0.0001) and for MIO-M1 cells was 53.1 ±
1.34 compared to the DMSO-equivalent of 89.95 ± 3.27 (P < 0.0001). At ½x, 1x, 5x, 10x of the
Brimonidine clinical dose, the mean cell viability for the ARPE-19 cells was 44.03 ± 0.74 (P <
0.0001), 46.07 ± 0.89 (P < 0.0001), 76.02 ± 1.18 (P < 0.0001), and 77.80 ± 1.05 (P < 0.0001),
respectively. For the MIO-M1, the cell viability values were 62.67 ± 0.41 (P < 0.0001), 64.15 ±
1.38 (P < 0.0001), 66.49 ± 0.64 (P < 0.0001), and 73.27 ± 1.42 (P < 0.0001) for the ½x, 1x, 5x and
10x Brimonidine doses, respectively. Cell viability for Brimonidine alone 10x and untreated cells
was 96.48 ± 0.77 and 97.56 ± 0.86 for ARPE-19 respectively; and 87.38 ± 0.49 and 97.41 ± 0.48 for
MIO-M1, respectively (Figures 1a and 1b).
Mitochondrial Membrane Potential (∆Ψm)
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The ∆Ψm values in the ARPE-19 cells exposed to 100 µM HQ were 43.8 ± 0.74 (P < 0.0001)
compared to 100µM DMSO-equivalent dose of 86.08 ± 0.39 and for MIO-M1 were 46.33 ± 1.07 (P <
0.0001) compared to the 100µM DMSO-equivalent of 86.55 ± 0.08. At ½x, 1x, 5x and 10x
Brimonidine co-treated groups, the ARPE-19 ∆Ψm values were 48.25 ± 0.58 (P = 0.0033), 49.13 ±
0.41 (P = 0.0008), 56.65 ± 0.58 (P < 0.0001) and 78.18 ± 0.90 (P < 0.0001), respectively (Figure 2a).
For MIO-M1 cells, only at 5x Brimonidine dose (56.05 ± 0.45 (P = 0.0001) and 10x Brimonidine
dose (77.85 ± 0.31 (P < 0.0001) was the ∆Ψm significantly reversed after treatment with HQ, (Figure
2b). ∆Ψm values for Brimonidine 10x alone and untreated cells were 83.4 ± 0.76 and 94.25 ± 1.38
respectively for ARPE-19 cells; and 82.80 ± 0.92 and 94.10 ± 0.69 respectively for MIO-M1 cells.
ROS/RNS Assay
ROS/RNS levels of the ARPE-19 cells were increased with 100 µM of HQ with relative
fluorescence values of 12400 ± 878. The ROS/RNS values were lowered after treatment with
Brimonidine at doses of 1x, (7944 ± 290, P = 0.041), at 5x dose (7830 ± 495, P = 0.045) and at 10x
dose (7887 ± 111, P = 0.0364), (Figure 3a). MIO-M1 cultures had increased ROS/RNS levels with
100 µM HQ (14300 ± 1078) and the ROS/RNS levels were decreased only at 10x dose Brimonidine
(8049 ± 72.5, P < 0.0286), (Figure 3b). The ROS/RNS levels for Brimonidine alone groups were
4159.5 ± 395 and 3410 ± 144 for ARPE-19 and MIO-M1 cells respectively. The results for 100 µM
DMSO groups were 4177 ± 156 for ARPE-19 cells and 4173 ± 152 for MIO-M1 cells. Untreated
ARPE-19 cells showed ROS/RNS levels of 3267 ± 46 and MIO-M1 cells of 3069 ± 43.5.
LDH Release Assay
ARPE-19 cells pretreated with 10x Brimonidine showed significant lower LDH levels compared to
100 µM HQ alone (1.6 ± 0.1 versus 2.63 ± 0.15, P = 0.0042). There was no significant protective
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effect for 1/2x Brimonidine (2.60 ± 0.058, P = 0.84), 1x dose (2.5 ± 0.058, P = 0.44) and 5x dose (2.5
± 0.065, P = 0.54), (Figure 4a). In the MIO-M1 cells, LDH levels did not decrease at any of the
studied concentrations: 2.516 ± 0.057 (P = 0.32), 2.554 ± 0.046 (P = 0.46), 2.494 ± 0.074 (P = 0.27)
and 2.479 ± 0.0337 (P = 0.11) for ½ x, 1x, 5x and 10x Brimonidine doses respectively, compared to
2.614 ± 0.0574 for 100 µM HQ-treated alone cells (Figure 4b). For ARPE-19 cells low LDH values
were found for Brimonidine 10x alone, 100 µM DMSO and untreated groups (0.57 ± 0.01, 0.57 ±
0.02 and 0.52 ± 0.007, respectively). Similar findings were observed for MIO-M1 cells with LDH
release of 0.73 ± 0.004, 0.73 ± 0.007 and 0.59 ± 0.032, for Brimonidine 10x alone, 100 µM DMSO
and untreated groups, respectively.
DISCUSSION
Previous studies have shown HQ toxicity in vitro in human RPE cells (ARPE-19), human Müller
retinal cells (MIO-M1), human microvascular endothelial cells (HMVEC) and rat neurosensory
retinal cells (R-28). 25, 26
In the present study the HQ at 100µM concentration caused more loss of cell
viability in the ARPE-19 cells compared to the MIO-M1 cells (27.92% versus 53.1%). In both cell
types the mechanisms involved were similar, reducing ∆Ψm, and increasing significantly the ROS
production and LDH release.
The goal of the study was to determine if Brimonidine might reverse the harmful effects of the HQ
exposure. Studies have shown that Brimonidine, apart from the intraocular pressure (IOP) lowering
effect, has a neuroprotective effect for glaucomatous neuropathy as well as other optic neuropathies
and injuries.5, 6, 8, 9
This is important because retinal ganglion cell loss is a key event in glaucomatous
neuropathy and vision loss; however, reducing the IOP is not enough to prevent the RGC loss. 4, 7, 8
In
addition, Brimonidine neuroprotective effects have been observed in vitro and in animal models, but
not in large controlled trials.7 Another factor that makes Brimonidine a possible cyto-protectant for
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the retina is that after topical instillation of Brimonidine, good concentrations have been found in the
retina 7, and both RPE and retinal Müller cells have alpha-2 adrenergic receptors.
11, 12, 14
Our study shows that Brimonidine improved CV of both cell lines and the Brimonidine protective
effects were dose dependent: the higher the Brimonidine dose, the higher the CV in both cell lines.
This effect was more pronounced in the ARPE-19 cells, where CV improved from 27.92% to 77.8%
in Brimonidine 10x group; while in the MIO-M1 the improvement was from 53.1% CV HQ alone to
73.27% at 10x. Also for ARPE-19 cells, the improvement in 10x group is higher than Brimonidine 5x
group but there is not statistically significant difference between them. Clinically, it is important to
identify cyto-protective drugs for human RPE cells as they are damaged in AMD, the most common
cause of vision loss in the elderly. In ARPE-19 cultures, the 10x dose Brimonidine increased CV by
50% compared to the HQ-treated alone. In addition, Brimonidine is a drug that has been used in
glaucoma patients for many years and has an excellent safety record. This makes Brimonidine an
attractive candidate for cyto-protection in early AMD or high risk patients.
While Brimonidine can protect both ARPE-19 and MIO-M1 cells, the mechanisms for action appear
to differ. In the ARPE-19 cells, all of the concentrations of Brimonidine improved the ∆Ψm in HQ-
treated cultures but in the MIO-M1 cells it was only the Brimonidine 5x and 10x concentration that
were protective. The 10x Brimonidine was the most effective concentration to increase the
mitochondrial membrane potential in both cell lines, and although it caused mild decrease in ∆Ψm
compared to DMSO control, the reversing effects to the HQ-induced toxicity were very significant. It
should be noted that even at 10x Brimonidine, the ∆Ψm values were still lower than in the DMSO
control.
Similar differential cyto-protective effects for Brimonidine were also found with respect to ROS
protection. Brimonidine significantly decreased ROS production on ARPE-19 cells at the 1x, 5x and
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10x concentrations but not in a dose-dependent fashion. In contrast, only the 10 x Brimonidine
concentrations were able to significantly reduce ROS production in the MIO-M1 cell line. These
findings suggest that the Brimonidine cyto-protective effects for the ARPE-19 cells are through an
oxidative stress pathway, which is generating ROS, while this pathway may be less involved in the
MIO-M1 cells. Alternatively, it may be that more ROS are produced by Müller cells than ARPE-19
cells and this necessitates higher doses of the drug to have a significant effect.
Increase in LDH release is typically an indicator of cell damage and necrosis. In our study, for both
ARPE-19 and MIO-M1 cells, HQ exposure resulted in significantly elevated levels of LDH, and only
the 10x Brimonidine significantly decreased LDH release for the ARPE-19 cells compared to HQ-
treated alone. Although there was a significant reduction of LDH levels in 10x Brimonidine group, it
was still 2.8 times higher than in DMSO control group. Interestingly, no changes in LDH levels were
found in MIO-M1 cells in any Brimonidine concentrations, suggesting that the necrosis pathway was
not involved. Studies have shown that in glaucoma neuropathy and induced optic nerve ischemia
models, apoptosis is responsible of the progressive RCG loss, and that Brimonidine can protect RCG
from that apoptosis process39-41
Previous studies in ARPE-19 and MIO-M1 cells have shown that HQ
did not induce apoptosis (no changes in caspase activities were shown), but in MIO-M1 cells,
autophagy markers were found.25, 26
In our study, Brimonidine treatment was nevertheless able to
increase the CV, suggesting that Brimonidine may be protecting ARPE-19 cells by blocking an
oxidative stress (ROS producing) pathway, while the cell viability of the MIO-M1 cells may be
protected via by a non-apoptosis/non-necrosis pathway (e.g. autophagy).
Our findings suggest that high Brimonidine doses (10x) can significantly block the HQ-induced
toxicity in immortalized human cell lines of RPE and retinal Müller cells. The Brimonidine can
improve the cell viability and mitochondrial membrane potential and also partially reduce the ROS
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formation in both cell lines. However, the Brimonidine had minimal to no effects on the LDH
production, which represents necrosis. Our findings indicate that (1) HQ-induced toxicity is mediated
through mitochondrial-damaging, oxidative stress-related and necrosis-related pathways; (2) while
Brimonidine can significantly reverse the mitochondrial-damaging and oxidative stress-related effects
in the ARPE-19 cells, other cyto-protective agents should be identified that can efficiently block the
necrosis component of HQ-toxicity; (3) the protective effects of Brimonidine differ between the
different retinal cell types (e.g., RPE cells versus Müller cells); and (4) human retinal cells can safely
be exposed to high concentrations of Brimonidine (10x) with minimal damaging effects.
In summary, these studies provide novel information regarding cell protection by Brimonidine in
human RPE cells and retinal Müller cells. The fact that Brimonidine can improve mitochondrial
health, reduce the oxidative stress damage, and confer cell protection, in in vitro and animal models
in RGC5-9, 13, 40, 42
, and now in RPE and Müller cells in vitro, opens the possibility of use for several
clinical entities. As mentioned above, RGC neuroprotection has not been shown yet in clinical trials 7,
43, but there are ongoing clinical trials using a Brimonidine implant for geographic atrophy, a final
irreversible stage in AMD. As AMD has such a large impact on vision loss in the aging populations,
more basic science research and clinical trials are needed to identify cyto-protective agents for this
disease, along with other age-related ocular disorders.
ACKNOWLEDGEMENTS
The present research was done with support by the Discovery Eye Foundation, Iris and B. Gerald
Cantor Foundation, Henry L. Guenther Foundation, Polly and Michael Smith Foundation, Lincy
Foundation.
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AUTHOR DISCLOSURES STATEMENT
Claudio Ramírez, Justin Chu, Joshua Chu, M. Tarek Moustafa, Marilyn Chwa, G. Astrid Limb and
M. Cristina Kenney have no financial interests to disclose related to the present research.
Javier Cáceres-del-Carpio is the David and Julianna Pyott Pan-American Retina Research Fellow and
has no financial interests to disclose related to the present research.
Baruch D. Kuppermann is a consultant to the following companies: AcuFocus, Alcon, Allegro,
Allergan, Ampio, Aqua Therapeutics, Genentech, Glaukos, Neurotech, Novartis, Ophthotech,
Regeneron, SecondSight, Staar Surgical, Teva, and Thrombogenics. Additionally, Dr. Kuppermann
has received grants/research support from Alcon, Allegro, Allergan, Genentech, Glaxo Smith Kline,
Neurotech, Ophthotech, Regeneron, ThromboGenics.
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REFERENCES
1. Arthur S, Cantor LB. Update on the role of alpha-agonists in glaucoma management. Exp Eye
Res. 2011; 93:271-283.
2. Toris CB, Gleason ML, Camras CB, Yablonski ME. Effects of brimonidine on aqueous
humor dynamics in human eyes. Arch Ophthalmol. 1995; 113:1514-1517.
3. Reitsamer HA, Posey M, Kiel JW. Effects of a topical alpha2 adrenergic agonist on ciliary
blood flow and aqueous production in rabbits. Exp Eye Res. 2006; 82:405-415.
4. Pfeiffer N, Lamparter J, Gericke A, Grus FH, Hoffmann EM, Wahl J. Neuroprotection of
medical IOP-lowering therapy. Cell Tissue Res. 2013; 353:245-251.
5. Weinreb RN. Is Neuroprotection a Viable Therapy for Glaucoma? Arch Ophthalmol. 1999;
117:1540.
6. Wheeler LA, Gil DW, WoldeMussie E. Role of alpha-2 adrenergic receptors in
neuroprotection and glaucoma. Surv Ophthalmol. 2001; 45 Suppl 3:S290-294; discussion
S295-296.
7. Saylor M, McLoon LK, Harrison AR, Lee MS. Experimental and clinical evidence for
brimonidine as an optic nerve and retinal neuroprotective agent: an evidence-based review.
Arch Ophthalmol. 2009; 127:402-406.
8. Bessero AC, Clarke PG. Neuroprotection for optic nerve disorders. Curr Opin Neurol. 2010;
23:10-15.
9. Danesh-Meyer HV. Neuroprotection in glaucoma: recent and future directions. Curr Opin
Ophthalmol. 2011; 22:78-86.
10. Ortin-Martinez A, Valiente-Soriano FJ, Garcia-Ayuso D, Alarcon-Martinez L, Jimenez-Lopez
M, Bernal-Garro JM, Nieto-Lopez L, Nadal-Nicolas FM, Villegas-Perez MP, Wheeler LA,
Vidal-Sanz M. A novel in vivo model of focal light emitting diode-induced cone-
Page 17 of 26
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Ocular Pharmacology and Therapeutics
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
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NLY/Not for DistributionRamírez, C., Cáceres-del-Carpio, J., et al
18
photoreceptor phototoxicity: neuroprotection afforded by brimonidine, BDNF, PEDF or
bFGF. PLoS One. 2014; 9:e113798.
11. Zhang N, Kannan R, Okamoto CT, Ryan SJ, Lee VH, Hinton DR. Characterization of
brimonidine transport in retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2006; 47:287-
294.
12. Harun-Or-Rashid M, Lindqvist N, Hallbook F. Transactivation of EGF receptors in chicken
Muller cells by alpha2A-adrenergic receptors stimulated by brimonidine. Invest Ophthalmol
Vis Sci. 2014; 55:3385-3394.
13. Semba K, Namekata K, Kimura A, Harada C, Mitamura Y, Harada T. Brimonidine prevents
neurodegeneration in a mouse model of normal tension glaucoma. Cell Death Dis. 2014;
5:e1341.
14. Vidal L, Diaz F, Villena A, Moreno M, Campos JG, Perez de Vargas I. Reaction of Muller
cells in an experimental rat model of increased intraocular pressure following timolol,
latanoprost and brimonidine. Brain Res Bull. 2010; 82:18-24.
15. Solberg Y, Rosner M, Belkin M. The association between cigarette smoking and ocular
diseases. Surv Ophthalmol. 1998; 42:535-547.
16. Group A-REDSR. Risk factors associated with age-related macular degeneration: A case-
control study in the age-related eye disease study: age-related eye disease study report number
3. Ophthalmology. 2000; 107:2224-2232.
17. Chew EY, Clemons TE, Agron E, Sperduto RD, Sangiovanni JP, Davis MD, Ferris FL, 3rd,
Age-Related Eye Disease Study Research G. Ten-year follow-up of age-related macular
degeneration in the age-related eye disease study: AREDS report no. 36. JAMA Ophthalmol.
2014; 132:272-277.
Page 18 of 26
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Ocular Pharmacology and Therapeutics
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review O
NLY/Not for DistributionRamírez, C., Cáceres-del-Carpio, J., et al
19
18. Cascella R, Ragazzo M, Strafella C, Missiroli F, Borgiani P, Angelucci F, Marsella LT,
Cusumano A, Novelli G, Ricci F, Giardina E. Age-related macular degeneration: insights into
inflammatory genes. J Ophthalmol. 2014; 2014:582842.
19. Chiras D, Kitsos G, Petersen MB, Skalidakis I, Kroupis C. Oxidative stress in dry age-related
macular degeneration and exfoliation syndrome. Crit Rev Clin Lab Sci. 2015; 52:12-27.
20. Talhout R, Schulz T, Florek E, van Benthem J, Wester P, Opperhuizen A. Hazardous
compounds in tobacco smoke. Int J Environ Res Public Health. 2011; 8:613-628.
21. Group WS. The Scientific Basis of Tobacco Product Regulation, Second Report of a WHO
Study GroupWHO Technical Repor Series 951. World Health Organization; 2008.
22. Pryor WA. Cigarette smoke radicals and the role of free radicals in chemical carcinogenicity.
Environ Health Perspect. 1997; 105 Suppl 4:875-882.
23. EPA. Hydroquinone Hazard Summary (Revised in January 2000). In: Agency EP, ed.
Environmental Protection Agency - Technology Transfer Network.
http://www.epa.gov/airtoxics/hlthef/hydroqui.html.
24. FDA. Hydroquinone Studies Under The National Toxicology Program (NTP).
25. Sharma A, Patil JA, Gramajo AL, Seigel GM, Kuppermann BD, Kenney CM. Effects of
hydroquinone on retinal and vascular cells in vitro. Indian J Ophthalmol. 2012; 60:189-193.
26. Ramirez C, Pham K, Franco MF, Chwa M, Limb A, Kuppermann BD, Kenney MC.
Hydroquinone induces oxidative and mitochondrial damage to human retinal Muller cells
(MIO-M1). Neurotoxicology. 2013; 39:102-108.
27. Sharma A, Neekhra A, Gramajo AL, Patil J, Chwa M, Kuppermann BD, Kenney MC. Effects
of Benzo(e)Pyrene, a toxic component of cigarette smoke, on human retinal pigment
epithelial cells in vitro. Invest Ophthalmol Vis Sci. 2008; 49:5111-5117.
Page 19 of 26
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28. Patil AJ, Gramajo AL, Sharma A, Chwa M, Seigel GM, Kuppermann BD, Kenney MC.
Effects of benzo(e)pyrene on the retinal neurosensory cells and human microvascular
endothelial cells in vitro. Curr Eye Res. 2009; 34:672-682.
29. Patil AJ, Gramajo AL, Sharma A, Seigel GM, Kuppermann BD, Kenney MC. Differential
effects of nicotine on retinal and vascular cells in vitro. Toxicology. 2009; 259:69-76.
30. Alcazar O, Cousins SW, Marin-Castano ME. MMP-14 and TIMP-2 overexpression protects
against hydroquinone-induced oxidant injury in RPE: implications for extracellular matrix
turnover. Invest Ophthalmol Vis Sci. 2007; 48:5662-5670.
31. Pons M, Marin-Castano ME. Cigarette smoke-related hydroquinone dysregulates MCP-1,
VEGF and PEDF expression in retinal pigment epithelium in vitro and in vivo. PLoS One.
2011; 6:e16722.
32. Pons M, Cousins SW, Csaky KG, Striker G, Marin-Castano ME. Cigarette smoke-related
hydroquinone induces filamentous actin reorganization and heat shock protein 27
phosphorylation through p38 and extracellular signal-regulated kinase 1/2 in retinal pigment
epithelium: implications for age-related macular degeneration. Am J Pathol. 2010; 177:1198-
1213.
33. Strauss O. The retinal pigment epithelium in visual function. Physiol Rev. 2005; 85:845-881.
34. Limb GA, Salt TE, Munro PM, Moss SE, Khaw PT. In vitro characterization of a
spontaneously immortalized human Muller cell line (MIO-M1). Invest Ophthalmol Vis Sci.
2002; 43:864-869.
35. Reichenbach A, Bringmann A. New functions of Muller cells. Glia. 2013; 61:651-678.
36. Franze K, Grosche J, Skatchkov SN, Schinkinger S, Foja C, Schild D, Uckermann O, Travis
K, Reichenbach A, Guck J. Muller cells are living optical fibers in the vertebrate retina. Proc
Natl Acad Sci U S A. 2007; 104:8287-8292.
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37. Garcia M, Vecino E. Role of Muller glia in neuroprotection and regeneration in the retina.
Histol Histopathol. 2003; 18:1205-1218.
38. Chwa M, Atilano SR, Reddy V, Jordan N, Kim DW, Kenney MC. Increased stress-induced
generation of reactive oxygen species and apoptosis in human keratoconus fibroblasts. Invest
Ophthalmol Vis Sci. 2006; 47:1902-1910.
39. Tatton WG, Chalmers-Redman RM, Tatton NA. Apoptosis and anti-apoptosis signalling in
glaucomatous retinopathy. Eur J Ophthalmol. 2001; 11 Suppl 2:S12-22.
40. Aktas Z, Gurelik G, Akyurek N, Onol M, Hasanreisoglu B. Neuroprotective effect of
topically applied brimonidine tartrate 0.2% in endothelin-1-induced optic nerve ischaemia
model. Clin Experiment Ophthalmol. 2007; 35:527-534.
41. Aktas Z, Gurelik G, Gocun PU, Akyurek N, Onol M, Hasanreisoglu B. Matrix
metalloproteinase-9 expression in retinal ganglion cell layer and effect of topically applied
brimonidine tartrate 0.2% therapy on this expression in an endothelin-1-induced optic nerve
ischemia model. Int Ophthalmol. 2010; 30:253-259.
42. Lee KYC, Nakayama M, Aihara M, Chen YN, Araie M. Brimonidine is neuroprotective
against glutamate-induced neurotoxicity, oxidative stress, and hypoxia in purified rat retinal
ganglion cells. Molecular Vision. 2010; 16:246-251.
43. Sena DF, Lindsley K. Neuroprotection for treatment of glaucoma in adults. Cochrane
Database Syst Rev. 2013; 2:CD006539.
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FIGURE LEGENDS
Figure 1: Cell viability assay (A) ARPE-19 cells treated with 1/2x, 1x, 5x and 10x Brimonidine and
exposed to HQ 100 µM. (B) MIO-M1 cells treated with 1/2x, 1x, 5x and 10x Brimonidine and
exposed to HQ 100 µM.
Figure 2: Mitochondrial membrane potential (∆Ψm) assay (A) ARPE-19 cells treated with 1/2x, 1x,
5x and 10x Brimonidine and exposed to HQ 100 µM. (B) MIO-M1 cells treated with 1/2x, 1x, 5x and
10x Brimonidine and exposed to HQ 100 µM.
Figure 3: ROS production (A) ARPE-19 cells treated with 1/2x, 1x, 5x and 10x Brimonidine and
exposed to HQ 100 µM. (B) MIO-M1cells treated with 1/2x, 1x, 5x and 10x Brimonidine and
exposed to HQ 100 µM.
Figure 4: LDH release (A) ARPE-19 cells treated with 1/2x, 1x, 5x and 10x Brimonidine and
exposed to HQ 100 µM. (B) MIO-M1cells treated with 1/2x, 1x, 5x and 10x Brimonidine and
exposed to HQ 100 µM.
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Cell viability assay (A) ARPE-19 cells treated with 1/2x, 1x, 5x and 10x Brimonidine and exposed to HQ 100
µM. (B) MIO-M1 cells treated with 1/2x, 1x, 5x and 10x Brimonidine and exposed to HQ 100 µM.
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Mitochondrial membrane potential (∆Ψm) assay (A) ARPE-19 cells treated with 1/2x, 1x, 5x and 10x
Brimonidine and exposed to HQ 100 µM. (B) MIO-M1 cells treated with 1/2x, 1x, 5x and 10x Brimonidine
and exposed to HQ 100 µM.
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ROS production (A) ARPE-19 cells treated with 1/2x, 1x, 5x and 10x Brimonidine and exposed to HQ 100
µM. (B) MIO-M1cells treated with 1/2x, 1x, 5x and 10x Brimonidine and exposed to HQ 100 µM.
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LDH release (A) ARPE-19 cells treated with 1/2x, 1x, 5x and 10x Brimonidine and exposed to HQ 100 µM.
(B) MIO-M1cells treated with 1/2x, 1x, 5x and 10x Brimonidine and exposed to HQ 100 µM.
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