Transcript Figure 1. The percent viability from the CFDA

on
Jennifer Winemiller, Department of Biological Sciences, York College of Pennsylvania
Abstract
Diabetes is caused by high levels of glucose circulating
through the body, also known as hyperglycemia. It has been
shown that accumulation of Advanced Glycation End Products
(AGEs) in the retina can lead to a loss of renal function. The
goal of this study is to determine the effect of AGEs on Retinal
Pigment Epithelial (RPE) cell viability through the use of
different AGE doses at 24 and 48 hours, and one week. A
CFDA-AM test was used to measure cell viability. At 24 and 48
hours, cell viability was reduced by about 50% at 100ug/ml.
During the one week trial, approximately 50% reduced viability
is noted at 0.001ug/ml. Throughout the study, all three
experiments showed a decrease in viability over time showing
that time had an effect on viability as well as AGE dose
concentration which seems to support my hypothesis.
Results
Methods
24 hour treatments
RPE-19 cells were divided in a 1:750 split into serum and serum free wells on a 96 well plate.
Cells were given AGE doses of 25, 50, 100, 250, and 500 µg/ml with each group containing 3
trials. After 24 hours they were treated with 50 µl CFDA-AM solution (Molecular Probe) and read
in the Wallac Plate Reader to test cell viability.
Discussion
At 48 hours RPE cells were divided in a 1:750 split into serum and serum free wells on a 96
well plate. Cells were given AGE doses of 25, 50, 100, 250, and 500 µg/ml with each group
having an containing 3 trials. After 48 hours they were treated with µl CFDA-AM solution and
read in the Wallac Plate Reader to test cell viability.
The 24 and 48 hour treatments both showed
decreases in cell viability of approximately 50% at 100
µg/ml. In another study, it was found that cell viability also
decreased drastically when treated with AGEs (Chibber
1997).
During the one week trial, the viability still decreased
by approximately 50%, but at a much lower dose. Only
0.001 µg/ml of AGEs were needed to obtain the reduction
in cell viability.
Throughout the study, all three experiments showed
a decrease in viability over time showing that AGE
treatments have both a dose and time dependent effect on
viability.
Using this data, we can conclude that AGEs, even at
a low dose over an extended period of time, can cause
decreases in cell viability which will eventually lead to
diabetic retinopathy in patients suffering from diabetes.
RPE cells were divided in a 1:30 split in serum free wells on a 6 well plate. Cells received
AGE doses of 0.001, 0.01, 0.1, 1,10, and 100 µg/ml. Cell viability was then measured after 7
days with 50 µl CFDA-AM solution and read in the Wallac Plate Reader to test cell viability.
Data Analysis
The values used in graphing were obtained by taking the average of the absorbances
measured by the Wallac plate reader and subtracting the background absorbance. Those
values were then divided by the percent control to produce an average percent viability with the
control representing 100%.
140
Serum Free
200
Serum
AGE-SF
AGE-Serum
Taxol-SF
Taxol-Serum
175
120
Literature Cited
100
Percent survival
Percent Viability
150
80
60
125
Chibber, R., Molinatti, P.A., Rosatto, N., Lambourne, B., and Kohner, E. M. 1997. Toxic action of
advanced glycation end products on cultured retinal capillary pericytes and endothelial cells: relevance
to diabetic retinopathy. Diabetologia. 40: 156-164.
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75
Lerman, O., Galiano, R., Armou, M., Levine, J., and Gurtner, G.. 2003. Cellular Dysfunction in the
Diabetic Fibroblast. American Journal of Pathology 162:303-312.
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0
0
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500
0
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AGE Dose (ug/ml)
AGE Dose (ug/ml)
Figure 1. The percent viability from the CFDA-AM test of
serum free, and serum treated human RPE-19 cells after being
treated with different concentrations of AGEs for 24 hours. N=
3± SEM
Figure 2. The percent viability from the CFDA-AM test of serum
free and serum treated human RPE-19 cells after being treated with
different concentrations of AGEs for 48 hours. Also, the percent
viability of cells that survived a taxol treatment that acted as a
negative control. N= 3± SEM
Lu, M., Kuroki, M., Amano, S., Tolentine, M., Keough, K., Kim, I., and Bucala, R., 1998. Advanced
Glycation End Products Increase Retinal Vascular Endothelial Growth Factor Expression. The Journal of
Clinical Investigation 101:1219-1223.
Treins, C., Giorgetti-Peraldi, S., Murdaca, J., and Van Obberghen, E. 2001. Regulation of Vascular
Endothelial Growth Factor Expression by Advanced Glycation End Products. The Journal of Biological
Chemistry 276:43836-43841.
Wartenberg, M., Dönmez, F., Ling, F.C., Acker, H., Hescheler, J., and Sauer, H. 2001. Tumor-induced
Angiogenesis Studied in Confrontation Cultures of Multicellular Tumor Spheroids and Embryoid Bodies
Grown from Pluripotent Embryonic Stem Cells. The FASEB Journal 15: 995-1005.
Yamagishi, S., Inagaki, Y., Okamoto, T., Amano, S., Koga, K., Takeuchi, M., and Makita, Z.. 2002.
Advanced Glycation End Products-Induced Apoptosis and Overexpression of Vascular Endothelial
Growth Factor and Monocyte Chemoattractant Protein-1 in Human Cultured Mesangial Cells. The
Journal of Biological Chemistry 277: 20309-20315.
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Percent Survival
Diabetes is caused by high levels of glucose circulating
through the body, also known as hyperglycemia. With these
high levels, glucose can react with amino acids to produce
early glycation products. From there, these products can
undergo more complex reactions to irreversibly become
Advanced Glycation End Products (AGEs) (Yamagishi 2002).
It has been shown that accumulation of AGEs can lead to a
loss of renal function (Yamagishi 2002). As this loss advances,
diabetic retinopathy, which is the thickening and death of blood
vessels, eventually results in blindness.
One of the believed causes of diabetic retinopathy is the
release of angiogenic factors such as Vascular Endothelial
Growth Factor (VEGF) (Treins 2001). There has been
evidence that VEGF is controlled by hypoxia-inducible factor 1
(HIF 1), especially under hypoxic conditions (Wartenberg
2001). HIF 1 is a transcription factor made up of HIF 1α and
HIF 1β, also known as aryl hydrocarbon nuclear translocator
protein or ARNT. Under hypoxic conditions, HIF 1α will bind
with either ARNT or p53, a tumor suppresser gene. If there are
high levels of phosphorylation, HIF 1α reacts with ARNT
resulting in higher levels of VEGF, which ultimately leads to
angiogenesis. However, if there are lower levels of
phosphorylation, HIF 1α reacts with p53, resulting in apoptosis.
The dose at which this switch occurs has yet to be determined.
My hypothesis is that cell viability will decrease in a dose
and time dependent fashion.
At 24 hours, the serum free and serum cells had the
lowest viability at 100 µg/ml. Figure 1.
At 48 hours, the serum free cells had the lowest
viability at 1000µg/ml AGE. The serum cells had the
lowest viability at 250µg/ml. Figure 2.
The 7 day treatment showed the lowest viability to
be at 0.001 µg/ml. Figure 3.
48 hour treatment
One week treatment
Introduction
Image courtesy of the National Eye Institute
Image courtesy of the National Eye Institute
Dose Dependent Effect of Advanced Glycation End Products
Human Retinal Pigment Epithelial cell viability
100
90
Figure 3. The percent viability from the CFDA-AM test of
serum free human ARPE-19 cells after being treated with
different concentrations of AGEs for seven days.
80
70
60
50
0.001
0.01
0.1
1
AGE Dose (ug/ml)
10
100
Acknowledgements
I would like to thank Dr. Ron Kaltreider for all his time and knowledge throughout this process.