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Latanoprost therapy reduces the levels of TGF beta 1 and gelatinases
in the aqueous humour of patients with exfoliative glaucoma
Anastasios G.P. Konstas
a,
*
, George G. Koliakos
b
, Costas H. Karabatsas
c
, Panayiotis Liakos
d
,
Ursula Schlo
¨tzer-Schrehardt
e
, Nikolaos Georgiadis
a
, Robert Ritch
f
a
A University Department of Ophthalmology, AHEPA Hospital, Thessaloniki, Greece
b
Department of Biological Chemistry, Aristotle University, Thessaloniki, Greece
c
University Department of Ophthalmology, University of Thessaly, Larissa, Greece
d
Department of Biological Chemistry, University of Thessaly, Larissa, Greece
e
Department of Ophthalmology, University of Erlangen-Nu
¨rnberg, Erlangen, Germany
f
Departments of Ophthalmology, The New York Eye and Ear Infirmary, New York, NY and The New York Medical College, Valhalla, NY, USA
Received 27 February 2005; accepted in revised form 11 July 2005
Available online 22 August 2005
Abstract
The aim of the present study was to evaluate the effect of latanoprost monotherapy on the aqueous humour concentrations of TGF-b1,
MMP-2, TIMP-2, MMP-9 and gelatinolytic activity in patients treated for exfoliative glaucoma (XFG). Aqueous samples from 50 XFG
patients treated with latanoprost and 50 age-matched XFG patients treated with timolol were collected during phacoemulsification cataract
surgery. The concentrations of TGF-b1, MMP-2, TIMP-2, MMP-9 and gelatinase activity were determined by commercial immunoassays.
The mean active TGF-b1 concentration in the aqueous was significantly lower in XFG patients treated with latanoprost compared with those
treated with timolol (3.1G0.65 vs 13.4G1.5 pg ml
K1
); (PZ0.0014). The mean total MMP-2 concentration was lower in latanoprost treated
patients (31.75G3.8 vs 81.5G7.2 ng ml
K1
); (P!0.0001). The TIMP-2 concentration was also lower in XFG-latanoprost treated patients
(73.8G6.81 vs 101.28G7.29 ng ml
K1
); (PZ0.0096). Latanoprost monotherapy has a marked effect on the aqueous concentration of TGF-
b1, MMP-2 and TIMP-2 in XFG patients. A better understanding of its effect on the pathobiology of the disease may lead to its earlier use in
the disease process to prevent progression from XFS to XFG.
q2005 Elsevier Ltd. All rights reserved.
Keywords: exfoliative glaucoma; latanoprost; timolol; TGF beta; aqueous humour
Exfoliation syndrome (XFS) is characterized by excess
synthesis and progressive accumulation of extracellular
fibrillar material (Ritch et al., 2003). The precise aetiology
of this systemic matrix disorder remains unknown, but it is
thought that transforming growth factor b1 (TGF-ß1) causes
an imbalance of matrix degrading enzymes, matrix
metalloproteinases (MMPs) and their inhibitors, tissue
inhibitors of metalloproteinases (TIMPs), contributing to
the progressive accumulation of exfoliation material,
leading to elevated intraocular pressure and eventually to
the development of exfoliative glaucoma (XFG), one of the
leading causes of visual loss in elderly patients (Ritch and
Schlo
¨tzer-Schrehardt, 2001; Schlo
¨tzer-Schrehardt et al.,
2001, 2003; Ritch et al., 2003).
Latanoprost, increases uveoscleral outflow by stimulat-
ing extracellular matrix degradation in the ciliary body via
upregulation and/or activation of MMPs (Lindsey et al.,
1996; Gaton et al., 1999). Nothing is known, however, about
the effect of latanoprost on the concentration of TGF-ß1,
MMPs and related molecules in the aqueous of XFS and
XFG patients. Therefore, we measured the aqueous
concentrations of TGF-b1, MMP-2, TIMP-2, and MMP-9
as well as the gelatinolytic activity in the aqueous from
patients with XFG receiving chronic latanoprost mono-
therapy, as compared to control XFG patients receiving
chronic timolol maleate monotherapy.
Experimental Eye Research 82 (2006) 319–322
www.elsevier.com/locate/yexer
0014-4835/$ - see front matter q2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.exer.2005.07.004
*
Corresponding author. Address: Associate Professor Anastasios G. P.
Konstas, MD, PhD, A University Department of Ophthalmology, AHEPA
Hospital, 1 Kyriakidi Street, Thessaloniki 546 36, Greece.
E-mail addresses: konstas@med.auth.gr (A.G.P. Konstas), koliakos@
med.auth.gr (G.G. Koliakos).
1. Patients and methods
1.1. Sample collection
Aqueous humour samples were collected during routine
phacoemulcification from patients with early/mild XFG
(defined arbitrarily as vertical cupping less than 0.75 and
mean visual field defect less than 10 dB) clinically
controlled on either latanoprost 0.005% (Xalatan, Pfizer
Ophthalmics, Athens, Greece), or timolol maleate 0.5%
(Temserin 0.5%, MSD/Vianex, Athens, Greece) monother-
apy. All patients enrolled had been treated with these drugs
for more than 3 months and had a clinical diagnosis of XFG.
Glaucoma was defined on the basis of either glaucomatous
optic disc damage (neural rim thinning or notching,
saucerization, etc.), or glaucomatous visual field changes
(nasal step or paracentral, Seidel’s, or arcuate scotoma, etc.)
or both. Patients with XFG on multiple drugs, patients with
POAG or other glaucomas, and patients with other ocular
pathologies or systemic disorders that could influence the
results of this study were excluded, as were those with a
history of previous ocular or laser surgery.
All patients gave written informed consent. Patients
received a dilated comprehensive examination the day prior
to surgery using pupillary dilatation to confirm the presence
of exfoliation material.
Aqueous humour was aspirated prior to the introduction
of viscoelastic or anterior chamber maintainer in the
anterior chamber. A small amount of aqueous (10–40 ml)
was withdrawn through an ab-externo corneal paracentesis
site using a 27-gauge needle on a tuberculin syringe, with
special care to avoid blood contamination or iris touch. One
hundred aqueous samples, 50 from XFG patients treated
with latanoprost 0.005% monotherapy (mean age 73.9G6.5
years) and 50 age-matched XFG control patients treated
with timolol maleate 0.5% monotherapy (mean age 72.6G
8.2 years) were included in this study. All aqueous samples
were stored at K80 8C.
1.2. Enzyme immunoassays
The aqueous concentrations of active TGF-b1, MMP-2,
MMP-9 and TIMP-2 were assessed in 10 ml samples,
without pre-treatment using commercially available ‘sand-
wich’ enzyme immunoassay kits according to the manu-
facturers’ instructions (Bender MedSystems Diagnostics
GmbH, Vienna, Austria for TGF beta 1; Oncogene research
products, San Diego, CA, USA for MMP-2 and MMP-9 and
Quantikine; R&D Systems, Minneapolis, MN, USA- Cat N
o
DTM200 for TIMP-2). All measurements were performed
in duplicate.
1.3. Gelatinase activity
Gelatinase activity in the aqueous humour was assessed
with a commercially available activity assay kit (Chemicon
International Inc, Temecula, CA 92590, USA). A 2 hr
incubation time was employed with a 10 ml sample. The
final optical density of each sample was subtracted from the
optical density of a standard provided by the manufacturer
and expressed as a percentage.
1.4. Statistical evaluation
GraphPad InStat statistical package for Windows was
used. Data are expressed as meanGstandard deviation (SD).
The Kolmogorov-Smirnov test was used for normal
distribution fitting. The unpaired, two tailed Student t-test
was used for comparisons and the Pearson test for the
estimation of correlation between sets of data. The statistical
significance limit was set to P!0,05.
2. Results
The numerical data of the study are summarized in
Table 1.
A significantly lower active TGF-b1 concentration was
observed in the samples from those XFG patients treated
with latanoprost (PZ0.0014). In these patients, the mean
TGF-b1 concentration was 3.1G0.65 pg ml
K1
compared
with a mean concentration of 13.4G1.5 pg ml
K1
observed
in the timolol treated patients.
Total MMP-2 concentration was estimated in the
same samples. Total MMP-2 concentration was signifi-
cantly lower in the XFG patients treated with
latanoprost (mean 31.75G3.8 ng ml
K1
) than in those
treated with timolol (mean 81.5G7.2 ng ml
K1
); (P!
0.0001). A statistically significant correlation was
calculated between TGF-b1 and total MMP-2 concen-
trations in XFG patients treated with timolol (PZ0.032;
nZ30) but not in patients treated with latanoprost (PZ
0.214; nZ30).
In patients treated with latanoprost, the mean TIMP-2
concentration was significantly lower (mean 73.8G
6.81 ng ml
K1
) than that in the timolol treated group
(101.28G7.29 ng ml
K1
); (PZ0.0096). A statistically sig-
nificant correlation was calculated between TGF-b1 and
total TIMP concentrations in XFG patients treated with
timolol (PZ0.041; nZ20) but not in patients treated with
latanoprost (PZ0.318; nZ20).
The mean MMP-9 concentration was low in both groups
and there was no significant difference between the
latanoprost group (0.844G0.0025 ng ml
K1
) and the timolol
group (0.847G0.003 ng ml
K1
); (PZ0.392).
Considering gelatinase activity no statistically significant
difference was established between the timolol treated
group (8.0G1.3%) and the latanoprost treated group (7.7G
1.8%); (PZ0.454).
A.G.P. Konstas et al. / Experimental Eye Research 82 (2006) 319–322320
3. Discussion
Our research group has previously reported the concen-
trations of both TGF-ß1 and gelatinases in the aqueous of
patients with XFS and cataract patients without therapy
(Koliakos et al., 2001; Schlo
¨tzer-Schrehardt et al., 2001,
2003). In these studies, the aqueous concentration of active
TGF-ß1 has been shown to be significantly higher in XFS
patients (14.2G2.8 pg ml
K1
) compared with the control
cataract patients (3.9G2.5 pg ml
K1
). In the present study,
latanoprost therapy reduced the level of active TGF b1,
known to be elevated in eyes with XFS/XFG (Schlo
¨tzer-
Schrehardt et al., 2001; Koliakos et al., 2001) to normal
levels (3–5 pg ml
K1
). Thus, the values in the timolol treated
XFG group, corresponded closely with the values of the
untreated XFS patients, whereas the values in the
latanoprost treated XFG group were found to be reduced
to normal levels of cataract patients. It appears, therefore,
that latanoprost interrupts the positive feedback cycle of
TGF b1 accumulation. However, the mechanism of
reduction of TGF-ß concentration by latanoprost needs
further elucidation.
TGF b1 increases TIMP-2 and MMP-2 expression, as
documented for human fibroblasts in vitro (Overall et al.,
1991; Azuma et al., 1996; Seeland et al., 2002; Philipp et al.,
2005). Hypoxia, characteristic of XFS/XFG, (Helbig et al.,
1994) can further modulate the effect of TGF b1 on MMP-2
and MMP-9 expression (Saed et al., 2002). In addition, the
reduced aqueous levels of ascorbic acid in XFS/XFG eyes
(Koliakos et al., 2002) may also increase MMP-2, which is
down-regulated by increased ascorbic acid concentrations in
human amnion derived cells (Pfeffer et al., 1998).
We have previously reported that the aqueous levels of
total MMP-2 measured 55.5G22.0 ng ml
K1
in the cataract
group and 102.5G42.9 ng ml
K1
in the XFS group
(Schlo
¨tzer-Schrehardt et al., 2003). Again, in the present
study, the values in the timolol treated group largely
corresponded to those of the untreated XFS patients,
whereas the values in the latanoprost treated group were
reduced below normal levels. In addition, TIMP-2 was
previously measured at a concentration of 54.5G
15.8 ng ml
K1
in the aqueous humour of cataract patients
and 80.3G17.5 and 114.5G48.7 ng ml
K1
in patients with
XFS and XFG, respectively (Schlo
¨tzer-Schrehardt et al.,
2003). The TIMP-2 concentration in timolol treated XFG
patients in the current study was similar to that of treated
XFG group, whereas the value of latanoprost treated
patients corresponded to that of untreated XFS patients.
In tissue sections of monkey eyes treated with
latanoprost, the immunoreactivity for MMP-2 and MMP-3
was increased in the ciliary muscle, whereas the amount of
collagen types IV and VI was decreased (Ocklind, 1998;
Sagara et al., 1999). It was concluded that latanoprost may
modulate the extracellular matrix by upregulation of MMPs
and thus facilitate uveoscleral outflow. Similarly, increased
MMP-1, MMP-2, and MMP-3 immunoreactivity has been
observed in the iris root, ciliary muscle and adjacent sclera
of monkey eyes after treatment with PGF
2a
isopropylester
(Gaton et al., 2001). Treatment of explant organ cultures
with latanoprost results in marked upregulation of MMP-3
and MMP-9 and a mild induction of MMP-2 in the ciliary
body, but not the trabecular meshwork, or retinal pigment
epithelium (el Shabrawi et al., 2000). A study examining the
effect of latanoprost and timolol on MMP expression in the
rabbit conjunctiva showed a marked upregulation of MMP-
3 and a mild increase in TIMP-2 in the conjunctiva of
latanoprost treated eyes only (Mietz et al., 2001).
According to these data, increased concentrations of
MMP-2, MMP-9, and TIMP-2 and a higher gelatinase
activity would be expected to occur in the aqueous humour
from patients on latanoprost treatment. In contrast, we found
a significant reduction in aqueous MMP-2 and TIMP-2
levels after chronic latanoprost therapy in patients with
XFG, whereas the levels of total MMP-9 and the gelatinase
activity remained unaffected.
As documented in other tissues, an increased MMP-2
level can be attributed to the effect of TGF b1(Overall et al.,
1991; Azuma et al., 1996; Seeland et al., 2002; Saed et al.,
2002; Philipp et al., 2005). Therefore, the observed decrease
of MMP-2 concentration after latanoprost treatment is
compatible with the observed decrease in TGF b1
concentration. These findings indicate that the effect of
latanoprost in the human aqueous may differ from that
found in localized tissue studies. Due to the direction of the
flow of the aqueous the levels of MMP-2 in a certain locus
of the surrounding tissues may differ from the levels of the
enzyme found in the aqueous humour.
The interpretation of our results is limited by the fact that
the aqueous samples investigated were from cataract
patients with mild XFG. Although a study of more advanced
Table 1
Mean aqueous levels of the molecules measured in the study and differences between the two treatment groups
Molecule Latanoprost group Timolol group
nMean SD nMean SD P
TGF-b1 (pg ml
K1
) 30 3.1 0.65 30 13.4 1.5 0.0014
MMP-2 (ng ml
K1
) 30 31.75 3.8 30 81.5 7.2 !0.0001
MMP-9 (ng ml
K1
) 23 0.844 0.0025 22 0.847 0.003 0.392
TIMP-2 (ng ml
K1
) 20 73.8 6.81 20 101.28 7.29 0.0096
Gelatinase activity (%) 31 7.7 1.8 31 8.0 1.3 0.4548
A.G.P. Konstas et al. / Experimental Eye Research 82 (2006) 319–322 321
XFG would have been ideal, these patients would not have
been controlled on latanoprost, or timolol monotherapy.
It remains to be known if and how the duration of
latanoprost monotherapy influences the results. It also
remains to be resolved if timolol therapy influences the
balance of these molecules.
Further research is required to prove in the future if, in
addition to the IOP-lowering effect, latanoprost can actually
inhibit the development of XFG.
Acknowledgements
This research was supported by ENTER and PENED
grants (Greek Ministry of Development and E.U.) and grant
912-OPT-0091-149 from Pfizer Ophthalmics (New York,
USA) to Dr AGP Konstas. The authors acknowledge the
excellent technical assistance of Ms Melpomeni Christo-
phoridou and the help of Dr Ioannis Tersis and Ms Maria
Economopoulou with the aqueous samples.
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A.G.P. Konstas et al. / Experimental Eye Research 82 (2006) 319–322322
