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Hypoxia-induced retinal neovascularization plays a central role in the pathogenesis of diabetic retinopathy. This study aimed to investigate whether hypoxia leads to the release of nuclear high mobility group box 1 (HMGB1) peptides from cultured retinal pigment epithelial ARPE-19 cells, to determine the effect of HMGB1 on angiogenic cytokine production and elucidate the involved signaling pathways. A chemical hypoxia mimetic agent, cobalt chloride, induced SIRT1 downregulation, HMGB1 nucleocytoplasmic relocation and extracellular release from ARPE-19 cells, implicating its autocrine function. Resveratrol treatment significantly reduced secretion of HMGB1 from ARPE-19 cells exposed to hypoxia. Cell proliferation and cell cycle analyses demonstrated that exogenous HMGB1 caused significant growth suppression and G1 cell cycle arrest in ARPE-19 cells. Morphological observations showed that HMGB1 enhanced adhesion, but suppressed migration of ARPE-19 cells. More intriguingly, HMGB1 up-regulated expression of angiofibrogenic factors in ARPE-19 cells, including VEGF, bFGF, TGF-β2, and CTGF. Signal profiling characterization indicated that HMGB1 triggered hyperphosphorylation of Akt, p38 MAPK, and NF-κB, but not that of ERK, JNK, and Smad2, whereas inhibition of PI3K, MAPK, or NF-κB significantly attenuated the HMGB1-driven cytokine overproduction in ARPE-19 cells. Functional neutralization with anti-TLR4 and -RAGE antibodies confirmed that both receptors were involved in the cytokine overproduction. In conclusion, chemically-mimicked hypoxia induced nucleocytoplasmic relocation and release of HMGB1 peptides, which in turn up-regulated the production of angiofibrogenic factors in RPE cells, thereby contributing to the pathogenesis of hypoxia-associated diabetic retinopathies. Conversely, blockades of intraocular HMGB1 bioavailability or signal activation may prevent angiofibrogenesis in development of diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Methylglyoxal (MGO), a glycolytic metabolite, induces oxidative injury and apoptotic cell death that play a pathogenetic role in age-related macular degeneration (AMD). This study examined the impact of MGO on cell proliferation and autophagy flux in retinal pigment epithelium (RPE) ARPE-19 cells and elucidated the underlying mechanism. Short-term MGO exposure suppressed cell proliferation without induction of apoptotic cell death, increased production of reactive oxygen species, and potentiated H 2 O 2 -exhibited cytotoxicity in ARPE-19 cells. Conversely, pretreatment with N-acetylcysteine, a ROS scavenger, and aminoguanidine, an MGO blocker, prevented MGO-induced growth retardation. MGO significantly enhanced autophagy flux and increased intracellular accumulation of autophagosomes, which was functionally confirmed by addition of autophagy enhancer or inhibitors. Signaling kinetic observation indicated that MGO remarkably triggered phosphorylation of Akt, ERK1/2, p38 MAPK, and JNK1/2. Blockade of kinase activity demonstrated that the hyperphosphorylation of Akt, ERK1/2, JNK, and p38 MAPK were all involved in the MGO-enhanced autophagy and growth-arresting effect in ARPE-19 cells. Moreover, pretreatment with autophagic flux inhibitors including 3-methyladenine, bafilomycin A, and chloroquine effectively ameliorated MGO- but not H 2 O 2 -mediated ARPE-19 cytotoxicity. In conclusion, modulation of autophagy flux activity by using autophagic or kinase inhibitors may be an applicable modality to treat AMD. [ABSTRACT FROM AUTHOR]

Abstract: All-trans retinoic acid (atRA) has been reported to inhibit the proliferation of retinal pigment epithelial (RPE) cells and used in treatment of proliferative vitreoretinopathy (PVR) in animal model. This study aimed at examining the effectiveness of atRA in inhibiting the extracellular matrix (ECM) biosynthesis by RPE cells and the RPE cell-mediated collagen gel contraction. Cultured RPE cells were treated with atRA and the expression of four ECM proteins (collagen types I, III, IV and laminin β1) was profiled. The results indicated that atRA treatment up-regulated de novo synthesis of collagen type I, but decreased that of laminin β1 in a dose-dependent manner. Moreover, the effect of atRA on RPE cell contraction was evaluated by measuring the area of collagen gel where RPE cells populated. Treatment with atRA significantly inhibited RPE cell-mediated collagen gel contraction. Addition of exogenous laminin nonapeptide into gels promoted RPE cell contraction, while atRA reversed the laminin-enhanced contractility. atRA treatment significantly suppressed the gene expression of integrin β3 but not αV subunit, and effectively inhibited the tyrosine phosphorylation of integrin β3 at residue 747 in RPE cells grown on laminin-coated dish, suggesting that atRA may suppress the RPE contractility through either inhibiting integrin β3 expression or abrogating the integrin β3-mediated signaling. In conclusion, atRA pharmacologically possesses a tissue-remodeling capacity and inhibits contractility of RPE cells. Therefore, atRA might be potentially a therapeutic agent for certain ocular disorders such as PVR. [Copyright &y& Elsevier]