Abstract
purpose. To determine which components present in oxidized LDL are responsible for the cytotoxicity associated with its internalization by culture ARPE19 cells.
methods. ARPE19 cells were grown in 24-well and 96-well plates. Cell viability was measured by MTT and/or adenosine triphosphate (ATP) content. LDL was oxidized with Cu+2 and oxysterol content analyzed by a novel HPLC method.
results. OxLDL showed increased cytotoxicity with prolonged oxidation. Analysis of the oxLDL showed a predominance of the 7-oxygenated products, 7α-hydroxycholesterol (7αHCh), 7β-hydroxycholesterol (7βHCh), and 7-ketocholesterol (7kCh). Addition of these oxysterols to the ARPE19 cell in free form indicated that 7kCh is the most cytotoxic of the oxysterols but at physiologically unrealistic concentrations. Partitioning of individual oxysterols into nonoxidized LDL at concentrations similar to those found in the oxLDL also indicated that 7kCh is the most cytotoxic of the oxysterols. Transition metals are tightly bound by LDL and play an important role in the oxidation of LDL, but do not seem to enhance its cytotoxicity directly.
conclusions. Prolonged oxidation of LDL increases the levels of 7kCh due to further oxidation of 7αHCh and 7βHCh. The formation of 7KCh seems to be responsible for most of the cytotoxicity associated with oxLDL internalization in ARPE19 cells.
Our interest is in understanding the pathogenesis of age-related macular degeneration, the leading cause of blindness in elderly individuals.
1 This is a complex disease that involves the aging process as well as genetics and environmental factors. The accumulation of cholesterol in Bruch’s membrane as a process of aging
2 as well as the epidemiologic association to atherosclerosis
3 suggests a mechanistic relation between these two diseases. In atherosclerosis, the accumulation of low-density lipoproteins (LDL) in arteries and its subsequent oxidation and ingestion by macrophages is believed to be critical in the formation of atherosclerotic plaques.
4 The internalization of oxidized LDL (oxLDL) by macrophages leads to foam cell formation, and this process is thought to be one of the principle causes of atherosclerosis.
5 The cytotoxicity of oxLDL has also been reported in aortic endothelial cells
6 7 and retinal pigment epithelium (RPE) cells.
8 In aortic endothelial cells, oxLDL was found to increase the amount of reactive oxygen species
6 and reduce biological activity.
7 In RPE cells oxLDL inhibits phagocytosis of rod outer segment membranes.
8 The oxidation of the esterified and unesterified cholesterol within the LDL particle generates a series of cholesterol oxides known as oxysterols that have potent pharmacological activities.
9 10 These include the inhibition of cholesterol synthesis and the induction of apoptosis and necrosis in a variety of cells.
10 Direct oxysterol cytotoxicity has been demonstrated in several tissue culture cell systems.
10 These oxysterols are the main suspects in cytotoxicity and other adverse biological effects associated with oxLDL.
11
Our hypothesis is that, as humans age, a slow accumulation of cholesterol occurs in Bruch’s membrane and choriocapillaris under the macula
2 and then gradually oxidizes. As this material oxidizes it becomes increasingly more toxic impairing both RPE and scavenging macrophage function leading to inflammatory responses similar to those in atherosclerotic plaques.
5 6 7 This could lead to the formation of drusen deposits, which further stress the RPE and generate additional toxic substances. Macrophages also release VEGF in response to oxLDL internalization,
12 which may contribute to the choroidal neovascularization observed in some of the more severe cases of AMD.
In our accompanying study we have shown that rat RPE cells will internalize human rhodamine-labeled LDL and form deposits in Bruch’s membrane within 24 hours.
13 This internalization does not seem to occur homogenously throughout the retina, suggesting that the fenestrated choroidal endothelium may have some filtering capabilities, allowing LDL to enter some locations and not others.
13 This may explain why in humans cholesterol accumulation seems to be greater in the macula than in the peripheral retina.
2
In this study, we used a novel approach to study the oxysterol cytotoxicity by partitioning different oxysterols into nonoxidized LDL. This allowed us to avoid the complexity of a full LDL oxidation and to present each oxysterol individually to the RPE cells in LDL at physiologically relevant concentrations. We also examined the effects of transition metals on oxysterol cytotoxicity, since they can have both beneficial and detrimental effects on retinal cells.
14 The effects of zinc are particularly interesting, since this metal may play a beneficial role in slowing the progression of AMD.
15
For the purposes of this article and its companion,
13 the word “cytotoxicity” refers to the measurable nonviable cell fraction, as determined in our assays.
ARPE19 cells were purchased from American Type Culture Collection (Manassas, VA). hTERT-RPE1 cells were purchased from BD Biosciences-Clontech (Palo Alto, CA). hTERT cells are telomerase-immortalized human RPE cells. Both cell types were cultured in DMEM/F12 containing 10% fetal calf serum, 2 mM glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin.
In cytotoxicity, experiments cells were grown in 24- and/or 96-well plates in serum-containing medium until confluent. The cells were then changed to serum-free medium and treated with oxLDL and/or oxysterols at different concentrations and for different times (see figure legends for details).