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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

Background

Materials and Methods

Results for RPE cell VEGF secretion

Illustration of Annexin’s Apoptoic Pathwway

Funding and Acknowledgment

References

OPTIONALLOGO HERE

Age-related macular degeneration (AMD) is the leading cause of adult eye disease among the industrialized nations, particularly in the elderly 1. 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.

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

The Significance of RPE CellsWe 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.

1. Hyman L. Epidemiology of eye disease in the elderly. Eye (Lond) 1987; 1 (Pt 2):3302. 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,

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.

Normal PBMC

05

1015

20

ASA HA

Treatment

%A

po

pto

sis 0.325

0.65

0.975

Cont: No Tx

Normal PBMC

0102030405060

Blue Light Exposure No light exposure

% A

popt

osis

Aspirinized PBMC

0

1

2

3

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6

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8

ASA HA

Treatment

% A

po

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sis

0.325mg/dl

0.65mg/dl

0.975mg/dl

Control: NoTreatment

Aspirinized PBMC

0

5

10

15

20

25

30

Blue Light Exposure No light exposure

%A

po

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Flow Cytometry Results showing no apoptosis vs apoptosis

Results and ConclusionResults 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.

Thanks to the Illinois Society for the prevention of Blindness for grant funding and The Richard A. Perritt Charitable Foundation

RESULTS from PBMC based assays

Keyb=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.

Hippuric acidAspirin

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