Oxidative and Endoplasmic Reticulum Stresses Mediate Apoptosis Induced by Modified LDL in Human Retinal Muller Cells

PURPOSE. We previously showed that extravasated, modified LDL is implicated in pericyte loss in diabetic retinopathy (DR). Here, we investigate whether modified LDL induces apoptosis in retinal Muller glial cells. METHODS. Cultured human retinal Muller cells (MIO-M1) were treated with highly oxidized glycated LDL (HOG-LDL, 200 mg protein/L) or native LDL (N-LDL, 200 mg protein/L) for up to 24 hours with or without pretreatment with N-acetyl-cysteine (NAC, a blocker of oxidative stress) and 4-phenylbutyrate (4-PBA, a blocker of endoplasmic reticulum [ER] stress). Effects of HOG-LDL on cell viability, apoptosis, oxidative stress, and ER stress were assessed by cell viability, TUNEL, and Western blot assays. In separate experiments, Muller cells were treated with 7-ketocholesterol (7-KC, 5-20 mu M) or 4-hydroxynonenal (4-HNE, 5-40 mu M) for up to 24 hours. The same markers were measured. RESULTS. HOG-LDL induced apoptosis (decreased cell viability, increased TUNEL staining, increased expression of cleaved PARP, cleaved caspase-3, and BAX; decreased Bcl-2), oxidative stress (increased NOX4 and antioxidant enzymes, catalase, and superoxide dismutase 2), and ER stress (increased phospho-eIF2 alpha, KDEL, ATF6, and CHOP). Pretreatment with NAC or 4-PBA partially attenuated apoptosis. In addition. NAC attenuated activation of ER stress. Similar to HOG-LDL, 7KC, and 4HNE also induced apoptosis, oxidative stress, and ER stress. CONCLUSIONS. Our data suggest that extravasated, modified lipoproteins may be implicated in apoptotic Muller cell death, acting at least partially via enhanced levels of oxidative and ER stresses. They support our main hypothesis that, in addition to hyperglycemia, extravasated and oxidized LDL is an important insult to the diabetic retina. (Invest Ophthalmol Vis Sci. 2012; 53:4595-4604) DOI: 10.1167/iovs.12-9910