7-Ketocholesterol is present in lipid deposits in the primate retina: potential implication in the induction of VEGF and CNV formation.

Seven-ketocholesterol (7kCh) is a toxic cholesterol oxide found in high levels in atherosclerotic plaques1 and is suspected of being involved in foam cell formation in macrophages.2,3 In vivo, cholesterol and cholesteryl esters found in LDL deposits form 7kCh through the Fenton reaction.4 This oxidation is usually catalyzed by copper and iron ions,5 which are relatively abundant in atherosclerotic plaques.6 Cholesteryl esters (CEs) are particularly susceptible to copper oxidation,4,5 making the CE-rich LDL-derived deposits commonly found in capillaries and arteries highly susceptible to this form of oxidation. Another path for the formation of 7kCh is photooxidation, which has been well described in vitro7 and may play an important role in the skin and retina. Cholesterol can be readily photooxidized in the presence of a suitable photosensitizer to form a series of hydroperoxide intermediates8,9 that can further oxidize to 7kCh. Both pathways may be sources of 7kCh in the retina. Vascular endothelial growth factor (VEGF) is a member of a family of cytokines best known for its role in promoting angiogenesis, maintaining vascular permeability, promoting hematopoietic cell development, and wound healing.10 VEGF expression is known to be affected by hypoxia11 and inflammation-related events.12 The transcriptional regulation of VEGF is complex, and two key transcription factors are known to be involved in its regulation, hypoxia inducible factor (HIF)-1α and Sp1.13 VEGF is also known to be induced by oxidized low-density lipoprotein (oxLDL)14 and 7kCh.15 Hypoxia induces VEGF (and many other genes) through HIF-1α. HIF-1α is a tightly regulated transcription factor11 that, under normoxic conditions, is continuously synthesized and degraded.11,16 Under hypoxic conditions HIF-1α degradation is inhibited, which allows it to travel to the nucleus and to dimerize with its partner, HIF-1β, to induce transcription.16 A number of proteins and small molecules are known to regulate HIF-1 activity mainly by interacting with proteins involved in its degradation pathway.16 Reactive oxygen species (ROS) are also known to play an important role in the induction of HIF-1α through a mitochondria-mediated process.16,17 VEGF is also known to be induced via HIF-independent pathways,12 which may be related to hypoxia and/or inflammation depending on the circumstances. These are the Ras oncogene,18 the nuclear factor κB (NF-κB),19,20 and the liver X receptors (LXRs)-mediated pathways.21 Ras involvement was first demonstrated in HIF-1α−/− mice when “tumors” generated with K-RAS-transformed embryonic fibroblast cells maintained their angiogenesis.18 This suggested that K-RAS could compensate for the loss of HIF function and maintain VEGF induction. Additional studies have shown the involvement of the Ras pathways in VEGF induction.12 NF-κB is an important transcription factor that mediates hypoxic and inflammatory responses and has been shown to be pivotal in the induction of VEGF.19,20 The NF-κB activation of VEGF may be dependent on K-RAS or mitochondrial reactive oxygen species (ROS).12 The LXR pathway for the induction of VEGF is particularly interesting because oxysterols such as 7kCh are agonistic ligands for LXRs.22 Although 7kCh is not a particularly potent agonist for LXR,23 it may be able to induce VEGF if it accumulates in sufficient amounts. In this study we demonstrate that 7kCh is present in the primate retina and is localized mainly to Bruch’s membrane and the choriocapillaris associated with suspected lipoprotein deposits. We also demonstrate that 7kCh can induce VEGF expression in cultured RPE cells and vascular endothelial cells. More important, we demonstrate that 7kCh-mediated VEGF induction is HIF-1α independent and is partially regulated by LXRs.