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

Abstract

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 Müller glial cells. METHODS. Cultured human retinal Müller 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, Müller cells were treated with 7-ketocholesterol (7-KC, 5-20 lM) or 4-hydroxynonenal (4-HNE, 5-40 lM) 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-eIF2a, 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 Müller 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 In previous work, we proposed that in addition to hyperglycemia, extravasation of plasma lipoproteins through leaking blood retinal barriers (BRB) and their subsequent modification (glycation, oxidation) are important in the propagation of DR. 12-18 Several lines of evidence support this concept. Clinical studies indicate that dyslipidemia is associated with the severity of DR In particular, DR is positively associated with serum levels of LDL, apolipoprotein B (ApoB), and LDL particle concentration in type 1 diabetic patients. 13,[19][20][21] However, dyslipidemia in the absence of diabetes does not cause retinal injury, and we suggest that breakdown of the BRB is the critical factor. Using immunohistochemistry (for ApoB and oxidized LDL [ox-LDL]), we identified the presence of intraretinal modified LDL in type 2 diabetic patients who had not yet developed clinical DR, with larger amounts proportionate to disease severity in patients with clinical DR This staining initially surrounded the inner retinal capillaries. We also confirmed the absence of ApoB and ox-LDL in normal human retina. 16 In ex vivo studies, ox-LDL was associated with apoptotic figures in human diabetic retinas. 16 In more severe DR cases with proliferative D...

Immune complex formation in human diabetic retina enhances toxicity of oxidized LDL towards retinal capillary pericytes

“…Intra-retinal ox-LDL was associated with apoptosis, and in proliferative DR, with macrophage infi ltration ( 8 ). Consistent with a generalized retinal injury ( 18 ), in vitromodifi ed LDL was toxic toward Müller cells and RPEs as well as toward vascular cells (9)(10)(11)(12)(13)(14)(15). In the present work, CD36 and CD64 blockade inhibited apoptosis and improved mitochondrial function ( Fig.…”

“…Consistent with a general neurovascular retinal insult in DR ( 18 ), we also found evidence of a broad array of cytotoxic effects: "highly oxidized, glycated LDL" promoted oxidative stress, endoplasmic reticulum (ER) stress, infl ammation and abnormal gene expression, and decreased viability, not only in retinal vascular cells (endothelial cells, pericytes), but also in Müller glia and retinal pigment epithelial cells (RPEs) (9)(10)(11)(12)(13)(14)(15).…”

“…Native LDL (N-LDL) was isolated by sequential ultracentrifugation (density 1.019-1.063) of fresh plasma pooled from 4 to 6 fasted healthy volunteers; ox-LDL was prepared as before ( 12 ). For preparation of insoluble ox-LDL-ICs, human ox-LDL antibodies were isolated using a two-step protocol involving affi nity chromatography with immobilized protein G (Protein G-Sepharose 4 Fast Flow; Amersham-Pharmacia Biotech, Piscataway, NJ) and fractionation by affi nity chromatography in Sepharose-linked ox-LDL ( 22 ).…”

“…In brief, pericytes were trypsinized, washed twice with cold disease ( 7 ). We therefore hypothesized that the dominant effects of lipoproteins in DR occur not in plasma, but rather after extravasation through leaking blood-retina barriers (BRBs) (8)(9)(10)(11)(12)(13)(14)(15); the extent of this leakage may be more important than plasma concentrations of lipoproteins. We further hypothesized that extravasated intra-retinal lipoproteins undergo extensive modification (glycation, oxidation), rendering them cytotoxic ( 8 ).…”

Biomarkers in Diabetic Retinopathy

“…Lyons' team has shown that extravasated and modified LDL and immune-complexed oxLDL are present in the retina of diabetic patients. They have also demonstrated that glycated LDL and/or oxLDL exert cytotoxicity to cultured human retinal capillary endothelial cells [192], pericytes [166,[192][193][194][195], retinal pigment epithelium [165], and Muller glial cells [196]. Finally, these glycated LDL could be shown to cause changes in cell signaling, gene expression, cell apoptosis, and autophagy [165,192,193,195,196].…”