Abstract
Purpose::
The accumulation of low density lipoproteins (LDL) in Bruch membrane is an early event that is hypothesized to promote age-related macular degeneration (AMD), the most common cause of blindness in western societies.
Methods::
RPE cells were exposed to LDL or oxidatively modified LDLs (ox-LDL). Total RNA was extracted and subjected to the Affymetrix U133 Plus 2.0 gene chip and/or RT-qPCR. Cell extracts were analyzed by electrospray mass spectrometry or western analysis. To determine a functional impact of ox-LDL treatment, RPE cells were analyzed by TUNEL labeling. Human maculas with AMD were analyzed by immunohistochemistry.
Results::
Microarray analysis showed a physiological and pathological transcriptional response after LDL and ox-LDL treatment, respectively. LDL induced a downregulation of cholesterol biosynthesis genes while ox-LDL induced transcriptional alterations in genes related to inflammation including pentraxin-3, matrix expansion, lipid metabolism, and apoptosis. Electrospray mass spectrometry showed that ox-LDL, but not LDL induced large increases in sphingomyelin, ceramides, and cholesterol in RPE cells. With TUNEL labeling, ox-LDL induced 38% apoptosis compared to <1% after LDL or control (p<0.0001). Ox-LDL, but not LDL stimulated RPE cells to secrete pentraxin-3 into the culture medium. Immunohistochemistry of 12 AMD samples showed strong staining for pentraxin-3 in normal Bruch membrane, but light staining within basal deposits.
Conclusions::
The transcriptional, biochemical, and functional data support the hypothesis that modified LDLs are one trigger for initiating early events in the pathogenesis of AMD.
Keywords: retinal pigment epithelium • lipids • oxidation/oxidative or free radical damage