Transcript Dose Dependant Effect of Advanced Glycation End Products on

Abstract
Dose Dependant Effect of Advanced Glycation
End Products on Human ARPE-19 Cell Viability
Diabetic retinopathy, which results in thickening and death of the blood vessels, is a
disease that has been linked to high concentrations of circulating advanced glycation end
products (AGE). The mechanism of AGE in this condition is not fully understood, but recent
work has shown possible similar pathogenic events that are caused by Hypoxia Inducible
Factor (HIF-1α) activation resulting in angiogenesis and apoptosis. The goal of our study
was to determine the effect AGE concentration resulted in a change of human retinal cell
viability. We treated retinal cells in media with 0 to 500 µg/ml of AGE in serum free media
with or without 0.2% BSA, were treated for 24 hr. Cell viability was assessed using a
standard CFDA assay. We observed no difference between serum free and 0.2% BSA
groups. There was significant difference (p< 0.01) between non-treatment group with the
50 µg/ml and 500 µg/ml groups in 0.2% BSA. We observed a bi-phasic event with two
groups of cell death occurring in the 25 µg/ml to 50 µg/ml and post 100 µg/ml AGE
dosages. The data suggests a possible role of AGE dosages in the development of altered
retinal cell viability suggesting a role in diabetic retinopathy.
Duane C. Kline, Department of Biology
York College of Pennsylvania
Advanced Glycation End Products
Block blood flow in the basement membranes of blood vessels
Proposed AGE Activation of HIF-1 Alpha
Discussion
Introduction
Diabetes is a growing epidemic worldwide and across the United States (1,2). The World
Health Organization census figures in 1985 found 30 million reported cases of diabetes.
Currently it is estimated that 177 million cases worldwide and the prediction for 2025 has
the number of cases reaching 300 million (1). Diabetes is the 7th leading killer in the United
States and increases the incidence rates of other illnesses including retinopathy,
neuropathy, renal failure and heart disease (2). Diabetes results from inefficient utilization
of insulin that results in high levels of glucose circulating throughout the body, termed
hyperglycemia. These conditions result in an increase of glycation of bio-chemicals through
non enzymatic reaction with free sugars, DNA, lipids, and amino acids, creating advanced
glycation end products (3). AGEs have been recently linked to diabetic retinopathy as one
of the major contributors to the disease (4).
Chemicals were added to cells in 100 µl of incomplete media +/- 0.2% BSA. Cells were
treated with a 100 µl dosage of AGE (25, 50,100, 250, and 500 µg/ml) or taxol (100 nM) as
a positive control (n=4), then incubated for overnight.
Cell Viability Assay
After treatment, the cells were washed in 100 µl of PBS. 50 µl of the CFDA (25 mM)
solution was added to cells and incubated at 37C for 2 hr under complete darkness.
Viability was measured in the form of cell fluorescence, which was assessed using a Wallac
Victor2 plate reader using 496 nm excitation and 530 nm emission. The fluorescent values
were then converted to percent control of treatments verses untreated values. The following
statistical analyses were performed: 1-way ANOVA with a Kruskal-Wallis post-test, MannWhitney test, and T-test.
HIF-1 Alpha + p53
Induces VEGF Expression
Activation of p53
Angiogenesis
Apoptosis
We observed a bi-phasic trend in AGE treated +/- 0.2% BSA on cellular viability.
There was an initial decrease in viability (25-50 μg/ml), with a recovery to nontreatment control level viability (100 μg/ml), followed by a gradual decrease in
viability at increasing AGE concentrations (>100 μg/ml). Explanation of this trend
maybe best understood using elements known to diabetic retinopathy
pathophysiology. Diabetic retinopathy is characterized by initial cellular death,
which we may be observing in this study at lower doses. This could increase the
amount of the p53 tumor proteins resulting in apoptosis. The increase of cellular
loss triggers a proliferatory response, which would explain the recovery stage and
possible activation of VEGF. In the body, thickening of the basement layers have
been recorded in both human patients and rat models (3,11). We found that after
100 μg/ml the viability decreased, suggesting a second activation of p53 or
unspecific mechanisms due to increased AGE concentrations.
Due to limitations, we were not able to fully elucidate the role of AGE in diabetic
retinopathy in this study. In future studies, we hope to use the following tests to
aid in modification of our current model of AGE manifestation in retinal cells:
•Caspase-3 Activity to measures apoptosis
•VEGF Expression to demonstrate angiogenesis
SF
BSA
Taxol SF
Taxol BSA
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We predict increased Caspase-3 activity in the 25-50 μg/ml and 250-500 μg/ml,
because of Caspase-3 activity is a commitment step in the apoptotic cascade.
The Caspase-3 activity would be very low at the 50-100 μg/ml treatments, while
VEGF expression would be increased due to cells undergoing proliferation.
*
**
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50
*
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25
0
50
100
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250
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450
500
550
Taxol
AGE Dose (g/ml)
Figure 2. Dose Response of AGE on Human Retinal Cells. Cells were treated for 24 hr with
0-500 µg/ml AGE dosages in incomplete serum free (SF) media +/- 0.2% BSA (Bovine Serum
Albumin). Cells were washed with PBS and treated with CFDA and incubated at 37C for 2 hr.
Fluorences was measured using Wallac Victor 2 and converted to percent control of treatment
verses untreated. Results found signifacant are denoted * for p<.05 and ** p<.01 using 1 way
ANOVA with a Kruskal-Wallis post-test.
Results

The future of AGE research could change the methods for diagnosing AGE
induced diabetic retinopathy. Cellular samples could be taken from patients to see
the amount of AGE stress that has endured. Currently, AGEs are being used to
stiffen and strengthen tissues cultured for transplants. Researchers have
exploited the AGE properties by allowing tissues to be incubated in a glucose rich
cell media and allowed to grow for up to 10 weeks with positive results (12). AGE
research will be very beneficial to the future patients with a vast number of novel
technologies that can be created.
Literature Cited
0
Cell Culture
Chemical Treatments
HIF-1 Alpha + ARNT
100
Materials and Methods
Human ARPE-19 cells (ATCC) were maintained in a Dulbecco’s modified Eagle medium
/F12+10% fetal bovine serum (Gibco, BRL) at 37C at 5% CO2. Cells were split 1:750 from
a T-75, one day prior to treatment into 96-well plate (VWR). 18 hr prior to treatment, cells
were shifted to 100 µl incomplete media +/- 0.2% BSA.
High Concentration
125
Cell Viability
(% Untreated)
While many aspects of this pathway has been observed and characterized under hypoxia
conditions, little is know of the molecular effects of AGEs on this pathway. Signaling of the
correct pathway is due to hypo- or hyper- phosphorlation of HIF-1α. This concentration can
be dependent of available oxygen or the amount of AGE concentration. The goal of our
study was to determine the effect of increasing dose of AGE on the viability of retinal
cells. Based on diabetic retinopathy pathophysiology we predict that increased AGE
dosage will decrease cell viability. From this research, a concentration set point can be
obtained to further evaluate AGE interactions and explicate the mechanism of action of the
retinal eye cells.
Low Concentration
Figure 1. Proposed AGE pathway in retinal cells. At low concentrations of AGE, HIF-1α
(Hypoxia Inducible Factor) binds ARNT (aryl hydrocarbon receptor nuclear translocator)
inducing VEGF (vascular endothelial growth factor) which causes angiogenesis. At high
AGE concentration, HIF-1α binds to initiate p53, which causes apoptosis (5,7,8,9).
Retinopathy is an illness that causes damage to the small blood vessels located inside the
eye (Fig. 3). AGEs have been shown to localize in the blood vessels, vascular basement
membranes, and retinal pericytes within diabetic patients (3). The blood vessels undergo
stages of thickening, loss of vascular permeability, death, and re-growth that eventually lead
to blindness. When non-diabetic rats intravenously injected with exogenous AGEs their
eyes undergo similar pathogenesis of diabetic retinopathy, which resulted in a loss of
pericytes (4).
AGEs and hypoxic conditions have been documented to activate HIF-1α, a translation
factor that regulates genes involved in apoptotic or angiogentic responses(5,6,7). Through
the induction of VEGF (vascular endothelial growth factor) and p53 (tumor suppressor
protein), respectively. Figure 1 shows the low AGE concentration pathway activating HIF-1α
binds ARNT (aryl hydrocarbon receptor nuclear translocator) causing angiogenesis while,
HIF-1α binding to p53 tumor suppressor protein that initiates an apoptotic cascade at high
concentrations (5,7,8,9).
Figure 3. Progression of Diabetic Retinopathy (Left) A normal eye with retina
and blood vessels clearly depicted. (Center) shows an eye with increased
number and size of blood vessels. (Right) Eye that has hemorrhaged causing
blood to encase the eye (10).
No significant difference between AGE treatments in serum free media +/- 0.2% BSA.
 AGE concentration of 25 µg/ml in serum free and 250 µg/ml in 0.2% BSA were
significantly lower than non-treatment control groups (p<0.05). AGE concentrations of 50
µg/ml and 500 µg/ml in 0.2% BSA were significantly lower than non-treatment control
groups (p<0.01).
 Taxol treatments caused a significant reduction in viability from non-treatment control
cells. (p<0.05).
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Acknowledgements
I would like to thank the following professors for their assistance during my thesis research:
Ron Kaltreider, Ph.D., Mentor
Karl Kleiner, Ph. D.