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The Effects of Aspirin and its Metabolites on Peripheral Blood Mononuclear cells and Human Retinal Pigment Epithelial Cells – Implications in the
Pathophysiology of Age-Related Macular Degeneration.
Omer Iqbal1 MD, Wells Brambl1, Albara Ottman1 BS, Jeffrey Gaynes1,2, Daneyal Sayed, Daniel Kahn, Felipe De Alba MD1, Bruce Gaynes1 OD, Jawed
Fareed1 PhD, Charles Bouchard1 MD.
1Loyola University Health System, 2Department of Laboratory Medicine and Pathology, University of Minnesota
The Significance of RPE Cells
Abstract
Age-related macular degeneration is one of the leading causes of
blindness in the world. Recently, a large clinical trial reported that the
long-term use of aspirin was responsible for its occurrence. Aspirin is a
commonly used drug especially in the elderly for the prevention of heart
disease. Interestingly, it has been proved earlier that the effect of aspirin
on the mechanisms leading to age-related macular degeneration is
different from that of prevention of heart disease. In this study we
propose to determine the effects of aspirin and its toxic metabolites on
Peripheral Blood Mononuclear Cells(PBMC) and human retinal pigment
epithelial cells maintained in culture medium. The levels of biomarkers
such as Vascular Endothelial Growth Factor (VEGF), Noxa and other
markers of cell death will be determined. The initial phase of this study
will focus on the apoptotic effects of Aspirin and its metabolites on
peripheral blood mononuclear cells with irradiation of blue light, which is
has been shown to be an inducer of oxidative stress
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We selected Retinal Pigment Epithelial Cells (RPE) because of the
following reasons. In early embryonic development, RPE spontaneously
transdifferentiate into neural retinal tissue.13 REP plays a critical role in
the development and maintenance of adjacent photoreceptors. These
photoreceptors generate a number of reactive oxygen species when
illuminated with light and their proliferation is important step in the
pathogenesis of ocular disease such as proliferative retinopathy. It is
proposed that toxic metabolites of aspirin trigger the increased release
of reactive oxygen species and eventual choroidal neovascularization.
Normal PBMC
Normal PBMC
15
10
0.65
5
0
0.975
ASA
HA
Treatment
% Apoptosis
0.325
20
Cont: No Tx
60
50
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20
10
0
Blue Light Exposure
Aspirinized PBMC
No light exposure
Aspirinized PBMC
30
8
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25
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20
0 .3 2 5 mg/dl
5
0 .6 5 mg/dl
4
0 .9 7 5 mg/dl
%Apoptosis
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RESULTS from PBMC based assays
%Apoptosis
Age-related macular degeneration (AMD) is the leading cause of adult
eye disease among the industrialized nations, particularly in the elderly1.
There is a critical clinical unmet need in the treatment of this condition
essentially because of the lack of proper understanding of its
pathophysiology. Approximately US$98 Billion in direct health care costs
of visual impairment due to AMD is spent in the US, Canada, and Cuba,
while globally it is estimated to be US$255 billion2. AMD is described to
be a degenerative disease of the central portion of retina, known as the
macula, which results in loss of central vision. Since central vision is
required for several daily activities, it significantly impacts the functional
status and quality of life. In general, depending on the progression of the
disease, AMD is either characterized as dry type, or wet type. The
pathogenesis is currently poorly understood, however ischemia and
oxidative stress remain to be the key factors involved. The hypoxia then
results in the release of factors such as Vascular Endothelial Growth
Factor (VEGF), and inflammatory signals, which help the growth of new
and abnormal vessesls3. This is especially true when attempting to
explain the pathogenesis of wet type AMD, also known as choroidal
neovascularization since it is clinically observed that there is growth of
new and abnormal vessels into the subretinal space 4. In particular is the
release of VEGF due to retinal ischemia, resulting in weaker vessels that
grow behind the retina and under the macula. As a result of these weaker
walls, these vessels begin to leak blood and fluid, causing the macula to
swell, eventually leading to damage to the central vision of the eye.
Several other etiologies have been discussed as well, such the
complement pathway, single nucleotide polymorphisms, macrophages,
etc.3 In addition, there are also several risk factors for AMD, including,
age(>50)5,6, smoking7, family history8, and cardiovascular disease9. Most
recently, however, an article by Klein et al found that chronic use of
aspirin for at least 10 years increased the risk of neovascular AMD10. It
comments on how aspirin’s mechanism on retinal vessels may be
different than that involving cardioprotection. In fact, previous studies
have shown that aspirin enhances choroidal neovascularization11 and in
a laboratory model, increase vascular density12. Particularly, aspirin’s
effects of promoting growth of new vessels occurs in the presence of cell
injury, such as retinal ischemia. Although, it is known that aspirin
enhances choroidal neovascularization in the presence of cell injury, it is
not clear as to what causes cell injury or how retinal ischemia occurs. It
is proposed that aspirin and its toxic metabolites causes oxidative stress
and cell injury triggering choroidal neovascularization.
Results for RPE cell VEGF secretion
% Apoptosis
Background
15
Contr ol : No
Tr ea tment
3
10
2
1
5
0
ASA
HA
Tre at m e nt
0
Bl ue Li ght Expos ure
No l i ght expos ure
Flow Cytometry Results showing no apoptosis vs apoptosis
Key
Illustration of Annexin’s Apoptoic Pathwway
b=blue, r=red, d=no light
lda=low dose aspirin (0.925 mg/dL)
hda=high dose aspirin (0.325 mg/dL)
hip=hippuric acid (0.325 mg/dL)
p=.0012 when compared to no light + sham
Using an ordinary 1 way ANOVA.
Results and Conclusion
Cell Culture VEGF Standard Curve
2.5
y = 0.0021x + 0.0857
R² = 0.9986
2
OD (450nm)
OPTIONAL
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1.5
1
0.5
Materials and Methods
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pg/ml
ARPE-19 (ATCC® CRL-2302™) cells were kindly provided by Dr. Knepper. RPE cells
were cutlured in DMEM, 10% FBS, Penicillin, Streptomycin, Ciprofloxacin at 37C, %5
CO2. RPE Cells were placed tyrpsonized and placed in a 24 well plate in a total of 500
µL of DMEM. The cells were grown out out to >95% confluency and then media was
changed. Cells cultured in Blue light, red light, or darkness, were submitted to either
sham, 0.925 mg/dL of aspirin (high dose), 0.325 mg/dL aspirin (low dose), 0.325 mg/dL
hippuric acid. Light was generated using 2 red or 2 blue LED’s powered by 3v CR2032
batteries attached to a freezer box in which the 24 well plates where kept out of the dark.
The plates were incubated with with or without drugs in blue light, red light, or no light
overnight for 16 hours. The supernatants were harvested and VEGF was quantified using
R&D Systems, Inc. Quantikine ELISA Human VEGF Immunoassay kit per manufactures
directions. Total supernatant protein concentration was determined using modified Lowry
method. VEGF concentration was normalized to the total concentration before statistical
analysis Ordinary one way ANOVA using Dunnett’s multiple comparison test was
performed using GraphPad Prism.
Under an IRB approved protocol, blood was drawn from healthy volunteers who have
taken aspirin and those who have not taken aspirin and placed in citrated tubes.
Following 1:1 dilution in PBS, 20 mL of whole blood was overlayed onto 10mL of Ficoll
Lymphocyte Separation Medium and spun at 1600 RPM for 30 minutes without breaking.
Peripheral Blood Mononuclear Cells (PBMC) were recovered, pooled, washed, and
resuspened in RPMI-1640 growth media with 10% FBS . Recovered PBMCs were then
counted and aliquoted appropriately. 2x106cells were then taken from each sample of
pooled PBMCs and subject to three different final concentrations were obtained using
Aspirin, and Hippuric Acid(an Aspirin metabolite) at .325mg/dl, .65mg/dl, abd .975mg/dl,
and continuous blue light (~430nm) irradiation overnight at 370C. These samples are
then tested with flow cytometry using Annexin V, and Propidium Iodide.
1200
Results PBMC: In the normal person, when the normal blood is
subjected to blue light, there was a a 26.77% increase in apoptosis
compared to the control. There was a concentration dependent
response due to aspirin on apoptosis. In the aspirinated blood when
it was subjected to blue light, there was a 22.16% increase in
apoptosis compared to the control. A similar augmentation was
observed with the addition of blue light.
Conclusion: These results suggest that blue light and aspirin by
themselves causes apoptosis. However, aspirin in addition to blue
light causes augmentation in apoptotic rate compared(VEGF).
Apoptosis and VEGF expression are key factors involved in age
related macular degeneration. More volunteers will be evaluated to
increase the power of the study.
RESULTS RPE CELLS: Hippuric acid causes a statistically
significant increase in VEGF secretion in RPE cells exposed to blue
light or no light (p=0.0012). However, hippuric acid in the presence
of red light prevents the increase in VEGF secretion, bringing the
levels to control levels. Aspirin does not cause VEGF secretion.
Funding and Acknowledgment
Thanks to the Illinois Society for the prevention of Blindness for grant
funding and The Richard A. Perritt Charitable Foundation
References
Aspirin
Hippuric acid
1. Hyman L. Epidemiology of eye disease in the elderly. Eye (Lond) 1987; 1 (Pt 2):330
2. Access Economics, prepared for AMD Alliance International, The Global Economic Cost of Visual Impairment, March 16, 2010. Table i.
3. Ding X, Patel M, Chan CC: Molecular pathology of age-related macular degeneration. Prog Retin Eye Res 2009;28:1
4. Yang Z, Stratton C, Francis PJ, et al. Toll-like receptor 3 and geographic atrophy in age-related macular degeneration. N Engl J Med 2008;
359:1456.
5. Age-specific causes of bilateral visual impairment.Weih LM, VanNewkirk MR, McCarty CA, Taylor HRArch Ophthalmol. 2000;118(2):264.
6. Age-specific prevalence and causes of blindness and visual impairment in an older population: the Rotterdam Study. Klaver CC, Wolfs RC,
Vingerling JR, Hofman A, de Jong PTArch Ophthalmol. 1998;116(5):653.
7. Cigarette smoking, fish consumption, omega-3 fatty acid intake, and associations with age-related macular degeneration: the US Twin
Study of Age-Related Macular Degeneration.Seddon JM, George S, Rosner BArch Ophthalmol. 2006;124(7):995.
8. Risk of incident age-related eye diseases in people with an affected sibling : The Beaver Dam Eye Study.Klein BE, Klein R, Lee KE, Moore
EL,