Current Research at Aravind Eye Care System
Role of aldose reductase in retinal
pigment epithelium: An understanding
towards the pathogenesis of diabetic
retinopathy
Investigator
: S. Senthil Kumari,
Aravind Medical Research
Foundation, Madurai
Senior Research
Fellow
: R. Sharmila
Funding Source : Department of
Biotechnology (DBT) –
RGYI (2011 -2013)
Background and aims
With increasing global prevalence of diabetes
especially type 2, diabetic retinopathy (DR) is
one of the causes of vision impairment. Clinically,
DR is manifested as basement membrane
thickening, pericyte loss, microaneurysms,
increased permeability, exudate deposits, retinal
microinfarcts and new blood vessel formation.
Among the various pathogenic mechanisms
reported for DR, the polyol pathway is believed
to play a central role.
Aldose reductase (AR2, alcohol: NADP +
oxidoreductase, EC 1.1.1.21) is the first and
rate-limiting enzyme in the polyol pathway. It is
a two-step process in which glucose is converted
into sorbitol and eventually to fructose. Sorbitol
is an alcohol, polyhydroxylated, and strongly
hydrophilic, and therefore does not diffuse
readily through cell membranes and accumulates
intracellularly with possible osmotic consequences.
The fructose produced by the polyol pathway can
become phosphorylated to fructose-3-phosphate
which is broken down to 3-deoxyglucosone;
both compounds are powerful glycosylating
agents that enter in the formation of advanced
glycation end products (AGEs). Thus, activation
of the polyol pathway, by altering intracellular
tonicity, generating AGEs precursors, and
exposing cells to oxidative stress (perhaps through
decreased antioxidant defences and generation
of oxidant species) can initiate and multiply
several mechanisms of cellular damage. Despite
three decades of intense investigations, including
some clinical studies, the details of AR mediated
hyperglycemic injury remain unclear. In particular,
the mechanisms which control and regulate the
expression of AR gene and the catalytic activity
of AR protein remain poorly understood.
In case of Diabetic retinopathy, vascular
endothelial growth factor (VEGF) is a potent
angiogenic and vascular permeability factor and
is implicated in new blood vessel formation.
High levels of VEGF have been well documented
in vitreous of proliferative diabetic retinopathy
patients. However, the secretory profile of
angiogenic and anti-angiogenic factor in relation
to polyol pathway is poorly understood.
Therefore understanding the role of AR in
retinal pigment epithelium (RPE) and its secretion
of VEGF is very essential. This will provide more
insight into the interplay between RPE aldose
reductase system and VEGF secretion. The
objectives are:
• To study the effect of high glucose (25mM)
on expression of aldose reductase in ARPE-19
cells under normoxia by Real-time PCR.
• To evaluate the effect of high glucose (25mM)
on the secretion of VEGF in ARPE-19 cells
under normoxia and hypoxia.
Effect of high glucose (25 mM) on
expression of aldose Reductase in ARPE-19
cells under normoxia
ARPE-19 cells were grown in DMEM/F12
medium with 10% FBS in T25 flask at 5% CO2
till 70-80% confluent. Then the cells were serum
Vol. XIV, No.1, January - March 2014
starved for 3hrs and challenged with two different
concentrations of glucose: 5mM (normal) & 25
mM (High glucose) for 24 hrs.
ALR expression was quantified by SYBR green
chemistry. Post confluent culture of ARPE-19
cells in DMEM/F12 medium was used. Total
RNA was isolated using TRIzol Reagent (Sigma,
USA). cDNA template from total RNA was
synthesized using Superscript III cDNA synthesis
kit (Invitrogen, USA). The cycle parameters
consisted of an initial denature step of 95°C for
10 minutes followed by 40 cycles of 95°C for 15
seconds and 60°C for 60 seconds. The effect of
high glucose on the expression of AR is shown in
the figure. A 50 fold increased ALR expression
was observed in cells challenged with 25 mM as
compared to 5 mM glucose.
Effect of glucose on VEGF secretion in ARPE19 cells under normoxia
The VEGF levels of cell supernatant obtained
from ARPE-19 cells incubated with different
glucose concentration (5mM (normal) & 25mM
(High) under normoxia conditions were estimated
by Sandwich ELISA. The secretion of VEGF by
the ARPE-19 cells was quantified at 3hrs as well as
24 hrs. The 3 hr time point was used to evaluate
the secretion of VEGF upon brief exposure to high
Effect of high glucose (25 mM) on expression of
aldose Reductase in ARPE-19 cells under normoxia
21
glucose. The levels of VEGF secreted by ARPE-19
cells challenged with high glucose are shown in
the figure below.
VEGF was detected in cell soup as early as
3 hrs of culture in both low and high glucose
condition. At 24hrs, substantial increase was
observed and only a marginal increase was
observed in cells challenged with high glucose as
compared to normal glucose.
ARPE-19 cells incubated with high glucose
(25mM) showed 50 fold increases in ALR
expression. However, no significant increase was
observed in VEGF secretion at 24hrs. More than
95% of the cells were viable when the cells were
challenged with high glucose. Further studies
are underway to evaluate the effect of glucose
on VEGF secretion for extended time duration
and also the effect of Aldose Reductase inhibitors
on VEGF secretion by ARPE-19 cells under
hyperglycemia.
Study outcome
ARPE-19 cell line with high glucose could be
used as an in vitro model system to study the
involvement of aldose reductase in the pathogenesis
of hyperglycemia related complications in human
retinal pigment epithelium.
Effect of glucose on VEGF secretion in ARPE-19 cells
under normoxia